Methods and vectors for site-specific recombination

ABSTRACT

The present invention provides methods for site-specific recombination in a cell, as well as vectors which can be employed in such methods. The methods and vectors of the present invention can be used to obtain persistent gene expression in a cell and to modulate gene expression. 
     One preferred method according to the invention comprises contacting a cell with a vector comprising an origin of replication functional in mammalian cells located between first and second recombining sites located in parallel. Another preferred method comprises, in part, contacting a cell with a vector comprising first and second recombining sites in antiparallel orientations such that the vector is internalized by the cell. In both methods, the cell is further provided with a site-specific recombinase that effects recombination between the first and second recombining sites of the vector.

This application is a continuation of PCT/US96/14123, which was filed onAug. 27, 1996, as a continuation-in-part of U.S. patent application Ser.No. 08/522,684, which was filed on Sep. 1, 1995, and issued on Sep. 1,1998, as U.S. Pat. No. 5,801,030.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to methods for site-specific recombinationin a cell, as well as vectors which can be employed in such methods. Themethods and vectors of the present invention can be used to obtainpersistent gene expression in a cell and to modulate gene expression.

BACKGROUND OF THE INVENTION

The past few years have heralded a sharp increase in the availability ofnew approaches to gene therapy, and the number of diseases that appearpotentially amenable to treatment using so-called "therapeutic genes".Generally, a therapeutic gene is a gene that corrects or compensates foran underlying protein deficit or, alternately, that is capable ofdown-regulating a particular gene, or counteracting the negative effectsof its encoded product, in a given disease state or syndrome. Moreover,a therapeutic gene can be a gene that mediates cell killing, forinstance, in the gene therapy of cancer. A successful therapeuticoutcome using gene therapy hinges on the appropriate expression of thetherapeutic gene, and potentially its long-term persistence in the host.Whereas expression can typically be effectively controlled using variouscloning strategies that are well known to those skilled in the art,persistence (i.e., the long-term expression of the gene in the hostcell) has proven more elusive.

As a general approach toward obtaining prolonged gene expression,researchers employ as a vehicle for transfer of the therapeutic gene avector which demonstrates longevity in the host. Stability is maximizedwith use of a vector that integrates into the genome of the host,allowing for simultaneous integration of the therapeutic gene carried bythe vector. Along these lines, retrovirus, which as part of its lifecycle integrates into the host genome, has proven a useful tool.However, the use of retrovirus is not without its attendant problems.For instance, stable integration of a retroviral vector is confined totarget cells that are actively synthesizing DNA; its carrying capacityis limited due to the relatively small size of the vector; the vectorexhibits a lack of tissue tropism; and such a vector is rapidlyinactivated by antibodies when given systemically. As a consequence ofthese and other shortcomings which accompany the use of a retrovirus,many researchers have turned to adenovirus (Ad) as an alternate vectorfor gene therapy (see, e.g., Rosenfeld et al., Science, 252, 431-434(1991); Rosenfeld et al., Cell, 68, 143-155 (1992)).

Ads are nonenveloped, regular icosahedrons, 65-80 nanometers indiameter, that consist of an external capsid and an internal core(Ginsberg (ed.), The Adenoviruses, Plenum Press, NY (1984)). The Ad corecontains a linear, double-stranded DNA molecule. Two human serotypes,namely Ad2 and Ad5, have been studied intensively and have provided thebulk of available information about Ads. This information, as well asthe various useful properties of Ad (among other things,replication-deficient recombinant viruses are easily made and producedin large quantities using complementing cell lines, Ad is capable ofinfecting almost all cell types including terminally differentiated ornon-proliferative cells, and no malignancies have been associated withAd infection) have enabled the exploitation of Ad as an efficient genedelivery vector both in vivo and in vitro (see, e.g., Rosenfeld et al.(1991), supra; Rosenfeld et al. (1992), supra; Engelhardt et al., Hum.Gene Ther., 4, 79-769 (1993); Crystal et al., Nat. Genet., 8, 42-51(1994); Lemarchand et al., Circ. Res., 72, 1132-1138 (1993); Guzman etal., Circ. Res., 73, 1202-1207 (1993); Bajocchi et al., Nat. Genet., 3,229-234 (1993); Mastrangeli et al., J. Clin. Invest, 91, 225-234(1993)).

Despite these advantages of Ad vectors, the long-term expression of anadministered therapeutic gene has not satisfactorily been obtained usingAd as a gene transfer vehicle. First generation Ad vectors deleted theessential E1 region of the virus (Rosenfeld et al. (1992), supra;Boucher et al., Hum. Gene Ther., 5, 615-39 (1994)). Trans gene productfrom these viral vectors is detected for approximately two weeks in therodent model system before returning to background levels. Incomparison, in immune-suppressed mice, the length of the period duringwhich gene expression can be detected following infection issubstantially increased, as is the level of expression (Yang et al., J.Virol., 69, 2004-15 (1995); Yang et al., Proc. Natl. Acad. Sci., 10,4407-11 (1994)). These data suggest that immune surveillance isresponsible for the relatively poor performance of the first generationAd vectors. Moreover, the inability of Ad to be maintained in a cellintegrated into the host cell genome means that cells which express thetherapeutic gene encoded by the vector are ultimately lost from thecell.

Accordingly, many researchers working with Ad have sought alternativemeans of stabilizing recombinant vectors such that long-lived geneexpression can be obtained. One such means that has proven effective inother systems is that of maintaining the integrity of the transferredgene in the host in the form of an episome. An episome is anextrachromosomal genetic element that replicates independently of thehost cell genome. To this end, the capability of Epstein-Barr virus(EBV) to form an episome in its latent state has been usurped byresearchers as a means to generate episomes from various double-strandedDNA templates.

EBV is a human lymphotropic herpes virus which causes infectiousmononucleosis and is associated with at least two human cancers (Reismanet al., Molec. Cell. Biol., 5, 1822-1832 (1985)). EBV contains twoorigins of replication, oriLyt and oriP. During latency, EBV exists as aclosed circular double-stranded DNA molecule which employs oriP in itsreplication, and which is maintained at a copy number ranging from 10 to200 per cell (Young et al., Gene, 62, 171-185 (1988)). Plasmids thatharbor oriP can be maintained in cells that also express the nuclearantigen EBNA-1 (see, e.g., Yates et al., Nature, 313, 812-815 (1985);Jalanko et al., Biochemica et Biophysica Acta, 949, 206-212 (1988);Kioussis et al., EMBO J., 6, 355-361 (1987); Jalanko et al., Arch.Virol., 103, 157-166 (1988); Sugden et al., J. Virol. 63, 2644-2649(1989)). In the presence of EBNA-1, oriP permits plasmid replication ina variety of mammalian cells that EBV is incapable of infecting inculture (Reisman et al., supra; Yates et al., Proc. Natl. Acad Sci., 81,3806-3810 (1984)). The oriP origin contains two cis-acting elements thatare required for its activity (reviewed in Middleton et al., Advances inVirus Research, 40, 19-55 (1991)). The elements are separated by 1,000base pairs (bp), and both are composed of multiple degenerate copies ofa 30 bp segment. The first element, termed the family of repeats, or FR,contains 20 tandem 30 bp repeats. The second element is comprised of a114 bp segment that contains a 65 bp dyad symmetry element, or DS.

Mutagenesis studies reveal that the two regions of oriP functionvis-a-vis each other in an orientation- and distance-independent manner(Middleton et al., supra). Deletion of the intervening spacer region, oraddition to this region of more than 1,000 bp, does not affect thefunction of oriP. The FR element is known to function as an enhancer,but its role in replication remains unclear. Moreover, the 20 tandemrepeats comprising FR can be replaced by multiple copies of DS. As fewas eight of the 20 tandem repeats found in the FR enhancer aresufficient in short term assays for both enhancer activity and plasmidreplication.

EBNA-1 binds to the 30 bp repeats present in both elements of oriP. TheEBNA-1 protein is required for the initiation of DNA replication near DSwhich occurs once per cycle during the S phase of the EBV cell cycle(Middleton et, al., supra). A glycine-alanine repetitive sequence whichcomprises approximately 1/3 of the protein and a small region in thecarboxyl terminus of the protein is dispensable for plasmid replication(Yates et al. (1985), supra; Lupton et al., Mol. Cell. Biol., 5,2533-2542 (1985)). However, this sequence prevents the immune systemfrom detecting EBNA-1 and, consequently, would appear necessary forpersistence of EBV and EBV-derived vectors (Levitskaya et al., Nature,375, 685-688 (1995)).

The stability of episomes obtained using EBV is potentially limiting forlong-term persistence and expression of a therapeutic gene inasmuch asthere is at least some possibility of an error in partitioning newlysynthesized episomes between daughter cells, and in view of thepervasive negative selection pressure with respect to extrachromosomalgenetic elements. Integration of the episome into a nondeleterious locusin the genome, i.e., a safe haven for the transferred gene, wouldobviate this potential lack of stability. Along these lines,adeno-associated virus (AAV) demonstrates a unique ability to integratewith high frequency into human chromosome 19q13.3-qter (Kotin et al.,Human Gene Therapy, 5, 793-801 (1994)). This ability of AAV to integrateinto a defined and benign genomic site eliminates the risk ofinsertional mutagenesis due to inadvertent gene activation orinactivation that accompanies random insertion of DNA (Shelling et al.,Gene Therapy, 165-169 (1994)).

In terms of its general features, AAV is a human parvovirus that can bepropagated either as an integrated provirus, or by lytic infection(Muzyczka, Current Topics in Microbiol. and Immunol., 158, 97-129(1992)), and which has been employed as a vector for eukaryotic cells(see,. e.g., U.S. Pat. Nos. 4,797,368 and 5,173,414; Tratschin et al.,Mol. Cell. Biol., 4, 2072-2081 (1984); GenBank Database Accession NumberJ01901 or AA2C). The lytic phase of AAV infection requires theexpression of the Ad early gene products E1a, E1b, E2a, E4, and VA RNA(Kotin et al., supra). Latent infections are established by infection ofAAV in the absence of helper virus. Under these circumstances, AAVefficiently integrates into the cellular genome, and is maintained inthat state unless challenged with Ad.

Only two components of AAV are required for locus-directed integrationof a foreign gene into the human genome: the Rep proteins, and the AAVinverted terminal repeats (ITRs). The four Rep proteins are each encodedby the same gene, and are generated by alternative splicing of nascentmRNAs (Kyostio et al., J. Virol., 68, 2947-2957 (1994)). The two largerRep proteins, Rep78 and Rep68, bind the AAV ITRs and act asATP-dependent, sequence-specific endonucleases with helicase activity tounwind the region of the ITRs during AAV DNA replication (Holscher etal., J. Virol., 68, 7169-7177 (1994)). The smaller Rep proteins, Rep52and Rep40, appear essential for the accumulation of single-strandedprogeny genomes used in packaging the virus.

The AAV ITRs are located at each end of the genome. When the viral ITRsare single-stranded, they form T-shaped hairpin structures (Snyder etal., J. Virol., 67, 6096-6104 (1993)). The origin of replication,packaging, integration and excision signals are also found locatedwithin the region of the viral ITRs. Binding of Rep proteins to the AAVITRs is consistent with the essential role of this region inITR-dependent replication and locus-directed integration. In the absenceof Rep proteins, replication does not occur, and integration into thegenome appears to be a random event (Kotin et al., supra). The ITRs canfunction either in their native state or as cloned into a plasmid asdouble-stranded DNA. With appropriate application of Rep proteins (e.g.,by providing Rep proteins through coding sequences located in trans),foreign sequences incorporated between the ITRs can be excised from aplasmid, replicated and integrated into the host cell genome.

The implementation of the EBV strategy to generate an episome, or theAAV strategy to stabilize an episome or sequences carried by the episomethrough incorporation into a host cell genome, requires tight regulationof the relevant regulatory protein in each system (e.g., EBNA-1 for EBV,and Rep proteins for AAV). One approach that has been employed byresearchers for achieving tight regulation of gene expression is tocontrol expression of a gene or genetic sequence via a site-directedrecombination event. Two well studied systems that allow site-specificrecombination and have been used in a variety of applications are thephage P1 Cre/Lox system and the yeast Flp/Frt system.

The Cre and Flp proteins belong to the λ integrase family of DNArecombinases (reviewed in Kilby et al., TIG, 9, 413-421 (1993); Landy,Current Opinion in Genetics and Development, 3, 699-707 (1993); Argos etal., EMBO J., 5, 433-440 (1986)). The Cre and Flp recombinases showstriking similarities, both in terms of the types of reactions theycarry out and in the structure of their target sites and mechanism ofrecombination (see, e.g., Jayaram, TIBS, 19, 78-82 (1994); Lee et al.,J. Biolog. Chem., 270, 4042-4052 (1995); Whang et al., Molec. Cell.Biolog., 14, 7492-7498 (1994); Lee et al., EMBO J., 13, 5346-5354(1994); Abremski et al., J. Mol. Biol., 192, 17-26 (1986); Adams et al.,J. Mol. Biol., 226, 661-673 (1992)). For instance, the recombinationevent is independent of replication and exogenous energy sources such asATP, and functions on both supercoiled and linear DNA templates.

The Cre and Flp recombinases exert their effects by promotingrecombination between two of their target recombination sites, Lox andFrt, respectively. Both target sites are comprised of invertedpalindromes separated by an asymmetric sequence (see, e.g., Mack et al.,Nucleic Acids Research, 20, 4451-4455 (1992); Hoess et al., NucleicAcids Research, 14, 2287-2300 (1986); Kilby et al., supra). Theasymmetry provides directionality to the recombination event. Namely,recombination between target sites arranged in parallel (i.e., so-called"direct repeats") on the same linear DNA molecule results in excision ofthe intervening DNA sequence as a circular molecule (Kilby et al.,supra). Recombination between direct repeats on a circular DNA moleculeexcises the intervening DNA and generates two circular molecules. Incomparison, recombination between antiparallel sites (i.e., sites whichare in opposite orientation, or so-called "inverted repeats") on alinear or circular DNA molecule results in inversion of the internalsequence. Even though recombinase action can result in reciprocalexchange of regions distal to the target site when targets are presenton separate linear molecules, intramolecular recombination is favoredover intermolecular recombination.

Both the Cre/Lox and Flp/Frt recombination systems have been used for awide array of purposes. For instance, site-specific integration intoplant, insect, bacterial, yeast and mammalian chromosomes has beenreported (see, e.g., Sauer et al., Proc. Natl. Acad. Sci., 85, 5166-5170(1988); Fukushige et al., Proc. Natl. Acad. Sci., 89, 7905-7907 (1992);Baubonis et al., Nucleic Acids Research, 21, 2025-2029 (1993); Hasan etal., Gene, 150, 51-56 (1994); Golic et al., Cell, 59, 499-509 (1989);Sauer, Mol. Cell. Biolog., 7, 2087-2096 (1987); Sauer et al., Methods:Companion to Methods in Enzymol., 4, 143-149 (1992); Sauer et al., TheNew Biologist, 2, 441-449 (1990); Sauer et al., Nucleic Acids Res., 17,147-161 (1989); Qin et al., Proc. Natl. Acad. Sci., 91, 1706-1710(1994); Orban et al., Proc. Natl. Acad. Sci., 89, 6861-6865 (1992)).Eukaryotic viral vectors have been assembled, and inserted DNA has beenrecovered, using these systems (see, e.g., Sauer et al., Proc. Natl.Acad. Sci., 84, 9108-9112 (1987); Gage et al., J. Virol., 66, 5509-5515(1992); Holt et al., Gene, 133, 95-97 (1993); Peakman et al., NucleicAcids Res., 20, 495-500 (1992)). Specific deletions of chromosomalsequences and rearrangements have also been engineered, and excision offoreign DNA as a plasmid from λ vectors is presently possible (see,e.g., Barinaga, Science, 265, 27-28 (1994); Rossant et al., NatureMedicine, 1, 592-594 (1995); Sauer, Methods in Enzymol., 225, 890-900(1993); Sauer et al., Gene, 70, 331-341 (1988); Brunelli et al., Yeast,9, 1309-1318 (1993); InVitrogen (San Diego, Calif.) 1995 Catalog, 35;Clontech (Palo Alto, Calif.) 1995/1996 Catalog, 187-188). Cloningschemes have been generated so that recombination either reconstitutesor inactivates a functional transcription unit by either deletion orinversion of sequences between recombination sites (see, e.g., Odell etal., Plant Physiol., 106, 447-458 (1994); Gu et al., Cell, 73, 1155-1164(1993); Lakso et al., Proc. Natl. Acad. Sci., 89, 6232-6236 (1992);Fiering et al., Proc. Natl. Acad. Sci., 90, 8469-8473 (1973); O'Gormanet al., Science, 251, 1351-55 (1991); Jung et al., Science, 259, 984-987(1993)). Similarly, positive and negative strategies for selecting orscreening recombinants have been developed (see, e.g., Sauer et al., J.Mol. Biol., 223, 911-928 (1992)). The genes encoding the Cre or Flprecombinases have been provided in trans under the control of eitherconstitutive, inducible or developmentally-regulated promoters, orpurified recombinase has been introduced (see, e.g., Baubonis et al.,supra; Dang et al., Develop. Genet., 13, 367-375 (1992); Chou et al.,Genetics, 131, 643-653 (1992); Morris et al., Nucleic Acids Res., 19,5895-5900 (1991)). The use of the recombinant systems or componentsthereof in transgenic mice, plants and insects among others reveals thathosts express the recombinase genes with no apparent deleteriouseffects, thus confirming that the proteins are generally well-tolerated(see, e.g., Orbin et al., Proc. Natl. Acad. Sci., 89, 6861-6865 (1992)).

More recently, researchers have employed replication-deficient Advectors containing the phage P1 gene for Cre as a means of studyingintracellular Cre-mediated recombination (Anton et al., J. Virol., 69,4600-4606 (1995)). In these experiments, the Cre-expressing Ad vectorwas supplied to cells along with another Ad vector, in which the codingsequence of a reporter gene was separated from any promoter by anextraneous spacer sequence flanked by parallel Lox sites. Cre-mediatedrecombination resulted in excision of the spacer sequence, and a turningon of the formerly silent reporter gene. This approach would appear toallow for only the positive modulation of gene expression, and notstable gene expression, inasmuch as the gene switched on by therecombination event will be expressed only as long as thereplication-deficient Ad vector is maintained in the host cell.Moreover, there is a possibility of the reverse recombination reactionsimultaneously switching off the reporter gene and imposing an upperlimit on the expression level to be obtained, due to continuingproduction of Cre within the host cell (Anton et al., supra, page 4605).

Accordingly, there remains a need for expression systems in which thepotential of Ad and other gene therapy vectors can be more fullyrealized. The present invention seeks to provide such expressionsystems. In particular, it is an object of the present invention toprovide methods for site-specific recombination in a cell, and vectorswhich can be employed in such methods, which allow prolonged geneexpression as well as modulation of gene expression, and which overcomesome of the aforementioned problems inherent in prior gene expressionsystems. These and other objects and advantages of the presentinvention, and additional inventive features, will be apparent from thedescription of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to methods for site-specific recombinationin a cell, as well as vectors which can be employed in such methods. Themethods and vectors of the present invention can be used to obtainpersistent gene expression in a cell and to modulate gene expression.

More specifically, one preferred method according to the inventioncomprises the steps of: (a) contacting a cell with a vector comprisingan origin of replication functional in mammalian cells located betweenfirst and second recombining sites located in parallel, such that thevector is internalized by the cell, and (b) providing the cell with asite-specific recombinase that effects recombination between the firstand second recombining sites of the vector.

Another preferred method according to the invention comprises, in part,the steps of: (a) contacting a cell with a vector comprising first andsecond recombining sites in antiparallel orientations such that thevector is internalized by the cell, and (b) providing the cell with asite-specific recombinase that effects recombination between the firstand second recombining sites of the vector.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C are schematic diagrams depicting a vector with parallelrecombining sites useful in the context of the present invention beforedelivery to the host cell (FIG. 1A) and the truncated virus or circularstructure (FIG. 1B) and episome (FIG. 1C) resulting from theintracellular recombination event which occurs after delivery of thevector to the host cell in accordance with the invention. Either aclosed-circular vector (i.e., flanking plasmid or viral sequencesindicated by the stippled line) or a linear vector (i.e., flanking viralsequences indicated by the stippled boxes) can be employed for deliveryof the episome to the host cell. The region between and including FRS toSCS can be in either orientation with respect to the flanking vectorsequences. The arrows indicate the direction of transcription.Abbreviations: RS, recombining site; FRS, first recombining site; SRS,second recombining site; SA, splice acceptor site; SD, splice donorsite; FCS, first coding sequence; SCS, second coding sequence; P,promoter; PG, passenger gene; ori, origin of replication.

FIGS. 2A-2F are schematic diagrams depicting potential locations of theglycine-alanine (Gly Ala) repeats relative to the coding sequence (CS)and hormone binding domain (HBD). The glycine-alanine repeats similarlycan be employed in the absence of any hormone binding domain.

FIGS. 3A-3X are schematic diagrams depicting further applicationsaccording to the present invention of the parallel recombination method.The arrows indicate the direction of transcription. The stipplingindicates that the vector can be either linear or circular. Open carrotsindicate that the sequence specified can be present anywhere on thevector. Open bars within a defined region of the vector indicate theportion of the vector in which the sequence specified can be located.Abbreviations: RS, recombining site; FRS, first recombining site; SRS,second recombining site; SA, splice acceptor site; SD, splice donorsite; FCS, first coding sequence; SCS, second coding sequence; TCS,third coding sequence; P, promoter; SP, second promoter; PG, passengergene; N, origin of replication; ITR, inverted terminal repeat; Rep, Repprotein coding sequence.

FIG. 4 is a schematic diagram depicting a vector with antiparallelrecombining sites useful in the context of the present invention,particularly in the regulation of gene expression by site-directedrecombination without episomal excision. Either a closed-circular vector(i.e., flanking plasmid or viral sequences indicated by the stippledline) or a linear vector (i.e., flanking viral sequences indicated bythe stippled boxes) can be employed. The region between and includingFCS to SCS can be in either orientation with respect to the flankingvector sequences. The arrows indicate the direction of transcription.Abbreviations: FRS, first recombining site; SRS, second recombiningsite; SA, splice acceptor site; SD, splice donor site; FCS, first codingsequence; SCS, second coding sequence; P, promoter.

FIGS. 5A-5G are schematic diagrams depicting further applicationsaccording to the present invention of the antiparallel recombinationmethod. The arrows indicate the direction of transcription. Thestippling indicates that the vector can be either linear or circular.Abbreviations: RS, recombining site; FRS, first recombining site; SRS,second recombining site; SA, splice acceptor site; SD, splice donorsite; FCS, first coding sequence; SCS, second coding sequence; P,promoter.

FIG. 6 is a restriction map depicting plasmid pEOBspLx-Puro-CMVLx.

FIG. 7 is a restriction map depicting plasmid pEOBspLx-Puro-CMVLx-βglu.

FIG. 8 is a restriction map depicting plasmid pAdCMV Cre.

FIG. 9 is a restriction map depicting plasmid pAdCMVCreRSVOrf6.

FIG. 10 is a restriction map depicting plasmid pKSEBNAOriP(NR).

FIG. 11 is a restriction map depicting plasmid pKSEBNA(NS).

FIG. 12 is a restriction map depicting plasmid pAdCLxPyTagOri.

FIG. 13 is a restriction map depicting plasmid pAdEPrRlr.

FIG. 14 is a restriction map depicting linearized plasmid pCGE1E2ori.The plasmid typically exists in closed-circular form.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for obtaining persistentexpression of a gene in a host cell and vectors which can be employed insuch methods. The methods and vectors can also be employed to modulategene expression. The methods and vectors according to this inventioncouple in a novel fashion (1) the unique capabilities EBV and AAV (i.e.,the ability of EBV to generate episomes, and the ability of AAV tointegrate into the human genome), (2) the recombinase activities ofrecombination systems such as the Cre/Lox and Flp/Frt recombinationsystems, and (3) various desirable properties of Ad, and other similarviral and plasmid vectors.

Definitions

For ease of reference, the abbreviations and designations used herein todescribe the present inventive methods and vectors are set forth inTable 1.

                  TABLE 1                                                         ______________________________________                                        Abbreviations and Designations                                                ______________________________________                                        AAV         Adeno-associated virus                                            Ad          Adenovirus                                                        bp          Base pairs                                                        cDNA        Complementary DNA                                                 CMV         Cytomegalovirus                                                   Cre         Phage P1 site-specific recombinase                                DNA         Deoxyribonucleic acid                                             DS          Dyad symmetry element comprising one                                          element of oriP                                                   EBV         Epstein-Barr virus                                                EBNA-1      Protein which binds oriP and modulates                                        replication of EBV and EBV-derived                                            episomes                                                          FCS         First coding sequence                                             Flp         Target site recognized by the yeast Frt                                       protein                                                           FR          EBV family of repeats comprising one                                          element of oriP                                                   FRS         First recombining site                                            Frt         Yeast site-specific recombinase                                   HSV         Herpes simplex virus types I and II                               ITR         AAV inverted terminal repeats that contain                                    sequences that function as an origin of                                       replication                                                       kb          kilobase pairs                                                    Lox         Target site recognized by the phage P1 Cre                                    protein                                                           oriLyt      EBV origin of repiication involved in                                         replication in the lytic phase                                    oriP        EBV origin of replication involved in                                         replication in the latent state                                   P           Promoter                                                          PCR         Polymerase chain reaction                                         PG          Passenger gene                                                    Rep         Proteins which bind the ITRs that act in                                      replication and integration into the                                          genome of AAV and AAV-derived vectors                             RNA         Ribonucleic acid                                                  RS          Recombining site                                                  RSV         Rous sarcoma virus                                                SA          Splice acceptor site                                              SCS         Second coding sequence                                            SD          Splice donor site                                                 SRS         Second recombining site                                           SV40        Simian virus 40                                                   ______________________________________                                    

Also, according to the invention and as further defined herein, a"vector" is a molecule (e.g., virus or plasmid) that serves to transfercoding information to a host cell. An "origin of replication" is asequence on a vector or host cell chromosome that renders extragenomicelements (e.g., viruses or plasmids) capable of replicatingindependently of the host cell genome.

A "gene" is any nucleic acid sequence coding for a protein or a nascentRNA molecule. A "passenger gene" is any gene which is not typicallypresent in and is subcloned into a vector according to the presentinvention, and which upon introduction into a host cell is accompaniedby a discernible change in the intracellular environment (e.g., by anincreased level of deoxyribonucleic acid (DNA), ribonucleic acid (RNA),peptide, or protein, or by an altered rate of production or degradationthereof). A "gene product" is either an as yet untranslated RNA moleculetranscribed from a given gene or coding sequence (e.g., mRNA orantisense RNA) or the polypeptide chain (i.e., protein or peptide)translated from the mRNA molecule transcribed from the given gene orcoding sequence. Whereas a gene comprises coding sequences plus anynon-coding sequences, a "coding sequence" does not include anynon-coding (e.g., regulatory) DNA. A gene or coding sequence is"recombinant" if the sequence of bases along the molecule has beenaltered from the sequence in which the gene or coding sequence istypically found in nature, or if the sequence of bases is not typicallyfound in nature. According to this invention, a gene or coding sequencecan be wholly or partially synthetically made, can comprise genomic orcomplementary DNA (cDNA) sequences, and may be provided in the form ofeither DNA or cDNA.

Non-coding sequences or regulatory sequences include promoter sequences.A "promoter" is a DNA sequence that directs the binding of RNApolymerase and thereby promotes RNA synthesis. "Enhancers" arecis-acting elements of DNA that stimulate or inhibit transcription ofadjacent genes. An enhancer that inhibits transcription is also termed a"silencer". Enhancers differ from DNA-binding sites forsequence-specific DNA binding proteins found only in the promoter (whichare also termed "promoter elements") in that enhancers can function ineither orientation, and over distances of up to several kilobase pairs,even from a position downstream of a transcribed region.

According to this invention, "recombining sites" are comprised ofinverted palindromes separated by an asymmetric sequence at which asite-specific recombination reaction can occur. The present inventiondescribes preferred embodiments wherein recombining sites (e.g., firstand/or second recombining sites) preferably are employed. However, moreor less recombining sites (e.g., 1, or a multiplicity of sites, such as,for instance, 3, 4, 5, or 6 recombining sites) also can be employed.Moreover, different types of recombining sites can be used, e.g., acombination of two Lox sites with two Flp sites. A "recombinase" is aprotein which carries out recombination between particular recombiningsites.

Also according to the invention, a coding sequence is "operably linked"to a promoter when the promoter is capable of directing transcription ofthat coding sequence. A promoter "adjoins" a recombining site (i.e., afirst or second recombining site) when the promoter is capable ofexerting its effect on transcription through the region of therecombining site and upon sequences linked to the recombining site. Acoding sequence (i.e., a coding sequence or a second coding sequence)"adjoins" a recombining site (i.e., a first or second recombining site)when the coding sequence is at such a distance from the recombining sitethat it can be transcriptionally controlled by a promoter linked to therecombining site which exerts its effect through the region of therecombining site and upon the coding sequence. For this to occur,preferably the coding sequence is located within about 1000 bp of therecombining site, and even more preferably the coding sequence islocated within about 500 bp of the recombining site. However, in caseswhere the promoter is separated from the coding sequence it controls byan oppositely-oriented intervening coding sequence, preferably thedistance between the promoter and coding sequence it controls is about5000 bp or less, more preferably, about 2000 bp or less. A"polycistronic message" is a single mRNA from which more than onepeptide or protein is translated, as described further herein.

Methods for Site-Directed Recombination

The present invention provides two methods to effect site-specificrecombination in a cell. Both recombination methods involve contacting acell with a vector comprising first and second recombining sites suchthat the vector is internalized by the cell, and then providing the cellwith a site-specific recombinase that effects recombination between thefirst and second recombining sites of the vector. The firstsite-specific recombination method involves the use of a vector (i.e., a"parallel recombination vector") comprising first and second recombiningsites located in a parallel orientation, whereas the secondsite-specific recombination method involves the use of a vector (i.e.,an "antiparallel recombination vector") comprising the first and secondrecombining sites located in an antiparallel (or opposite) orientation.

The parallel recombination method is capable of effecting site-specificrecombination in a cell such that the recombination event generates anepisome comprising an origin of replication capable of functioning inmammalian cells, which can replicate autonomously of the host genome andcan be employed to impart stable maintenance in host cells upon vectorscarrying one or more passenger genes. The parallel recombination methodis useful in that it can be employed to stabilize vectors whichtypically do not integrate into the host cell genome (e.g., Ad or HSVvectors) by imparting to such vectors an ability to replicate via a"lysogenic-like" pathway. Thus, the method can be employed to obtainstable gene expression by stabilizing vectors carrying one or morepassenger genes.

The parallel recombination method also can be employed to effectsite-specific recombination in a cell such that the recombination eventgenerates a so-called "miniplasmid", which does not comprise an originof replication that is capable of functioning in mammalian cells, andwhich is not capable of replicating autonomously of the host genome.Notably, the second recombination product generated by the parallelrecombination event (i.e., the product other than the episome orminiplasmid, which itself comprises either a linear or a closed-circularstructure depending on whether the original substrate for therecombination reaction is, respectively, either linear orclosed-circular) also may be functional in the cell. Such a miniplasmid,as further described herein, can prove of use for the short-termdelivery of proteins (e.g., Rep protein) to cells.

Alternately, it also is possible that the vector employed forsite-specific recombination comprises more than one origin ofreplication prior to the recombination event. Vectors that comprise morethan one replication origin are known in the art.

As described further herein, both the parallel and antiparallelrecombination methods can be employed to either up- or down-regulatetranscription of a coding sequence, or to simultaneously up-regulatetranscription of one coding sequence and down-regulate transcription ofanother, through the recombination event. The antiparallel recombinationmethod differs from the parallel recombination method in that it doesnot involve the formation of an episome or miniplasmid.

The recombination methods according to the invention are in a cell,which preferably is a eukaryotic cell. A eukaryotic cell is a cell whichpossesses a true nucleus surrounded by a nuclear membrane. Preferablythe eukaryotic cell is of a multicellular species (e.g., as opposed to aunicellular yeast cell), and even more preferably is a mammalian(optimally human) cell. However, the methods also can be effectivelycarried out using a wide variety of different cell types such as avianand fish cells, and mammalian cells including but not limited to rodent,ape, chimpanzee, feline, canine, ungulate (such as ruminant or swine),as well as human cells. Moreover, if vector transfer to a particularcell type is limited due, for instance, to a lack of receptors for aparticular virus such as adenovirus, transfer can be increased usingmethods employed, for example, to carry human adenovirus into blood orother cell types. For instance, the virus can be coupled to aDNA-polylysine complex containing a ligand (e.g., transferrin) formammalian cells (Wagner et al., Proc. Natl. Acad. Sci., 89, 6099-6103(1992)), or by using other similar methods which are known to thoseskilled in the art.

Any suitable vector can be utilized in the present inventive methods,particularly the novel vectors described herein. Thus, the vectorutilized in accordance with the present inventive methods can encompassany vector, linear or circular, that is appropriate for introduction ofnucleic acids into eukaryotic cells, and is capable of functioning as avector as that term is understood by those of ordinary skill in the art,so long as the vector is predominantly comprised of double-stranded DNAduring some phase of its existence. Preferably, the resultant vector iscompatible with the host cell, i.e., is capable of transcribing thenucleic acid sequences subcloned in the vector, and if desired, also iscapable of translating the nascent mRNA.

The vector optimally is of viral origin, and can contain one or moreheterologous or recombinant sequences, e.g., a coding sequence, apassenger gene, promoter, or the like. Preferably the vector containsthe minimal sequences required for packaging and delivery to the cell.The vector desirably is comprised, in part, of a virus that is eitherenveloped or nonenveloped. For instance, preferably a vector is anonenveloped virus from the family Hepadnaviridae, Parvoviridae,Papovaviridae, or Adenoviridae. A preferred nonenveloped virus accordingto the invention is a virus of the family Hepadnaviridae, especially ofthe genus Hepadnavirus. A virus of the family Parvoviridae desirably isof the genus Parvovirus (e.g., parvoviruses of mammals and birds) orDependovirus (e.g., adeno-associated viruses (AAVs)). A virus of thefamily Papovaviridae preferably is of the subfamily Papillomavirinae(e.g., the papillomaviruses including, but not limited to, humanpapillomaviruses (HPV) 1-48 and bovine papillomaviruses (BPV)) or thesubfamily Polyomavirinae (e.g., the polyomaviruses including, but notlimited to, JC, SV40, BK virus, and mouse polyomavirus). A virus of thefamily Adenoviridae desirably is of the genus Mastadenovirus (e.g.,mammalian adenoviruses) or Aviadenovirus (e.g., avian adenoviruses).Similarly, a vector can be an enveloped virus from the familyHexpesviridae, or can be a Sindbis virus. A preferred enveloped virusaccording to the invention is a virus of the family Herpesviridae,especially of the subfamily Alphahezpesvirinae (e.g., the herpessimplex-like viruses), genus Simplexvirus (e.g., herpes simplex-likeviruses), genus Varicellavirus (e.g., varicella and pseudorabies-likeviruses), subfamily Betaherpesvirinae (e.g., the cytomegaloviruses),genus Cytomegalovirus (e.g., the human cytomegaloviruses), subfamilyGammaherpesvirinae (e.g., the lymphocyte-associated viruses), or genusLymphocryptovirus (e.g., EB-like viruses). In particular, preferably thevirus component of the vector is selected from the group consisting ofAd, herpes simplex virus types I and II (HSV), EBV, vaccinia virus,papilloma virus (e.g., either human (HPV) or bovine (BPV)), JC, simianvirus 40 (SV40), polyomavirus (e.g., either human or mouse), hepatitisvirus B, and cytomegalovirus (CMV). Moreover, for practice of theparallel recombination method wherein an episome desirably is formed,preferably, the vector is comprised, in part, of vectors derived fromviruses which do not form proviruses as part of their replicative cycle.

An especially preferred vector according to the invention is anadenoviral vector (i.e., a viral vector of the family Adenoviridae,optimally of the genus Mastadenovirus). The adenovirus is of anyserotype vector. Adenoviral stocks that can be employed as a source ofadenovirus can be amplified from the adenovirus serotypes from type 1through 47 currently available from American Type Culture Collection(ATCC, Rockville, Md.), or from any other serotype of adenovirusavailable from any other source. For instance, an adenovirus can be ofsubgroup A (e.g., serotypes 12, 18, 31), subgroup B (e.g., serotypes 3,7, 11, 14, 16, 21, 34, 35), subgroup C (e.g., serotypes 1, 2, 5, 6),subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33,36-39, 42-47), subgroup E (serotype 4), subgroup F (serotype 40, 41), orany other adenoviral serotype. Preferably, however, an adenovirus is anAd2 or Ad5 serotype.

The Ad employed for nucleic acid transfer can be wild-type (i.e.,replication-competent). Alternately, the Ad can comprise geneticmaterial with at least one modification therein, which can render thevirus replication-deficient. The modification to the Ad genome caninclude, but is not limited to, addition of a DNA segment, rearrangementof a DNA segment, deletion of a DNA segment, replacement of a DNAsegment, or introduction of a DNA lesion. A DNA segment can be as smallas one nucleotide and as large as 36 kilobase pairs (kb) (i.e., theapproximate size of the Ad genome) or, alternately, can equal themaximum amount which can be packaged into an Ad virion (i.e., about 38kb). Preferred modifications to the Ad genome include modifications inthe E1, E2, E3 or E4 region.

In the methods and vectors of the present invention, the vectors mayfurther comprise a passenger gene. The passenger gene can be in anyorientation in the vector. Any suitable passenger gene can be employed,such as a reporter gene or a therapeutic gene, so long as the passengergene is capable of being expressed in a cell in which the vector hasbeen internalized. For instance, the gene can comprise a reporter gene,or a nucleic acid sequence which encodes a protein that can in somefashion be detected in a cell. The gene also can comprise a therapeuticgene which exerts its effect at the level of RNA or protein. Forinstance, the protein can be employed in the treatment of an inheriteddisease, such as, e.g., the cystic fibrosis transmembrane conductanceregulator cDNA for the treatment of cystic fibrosis. The protein encodedby the therapeutic gene may exert its therapeutic effect by resulting incell killing. For instance, expression of the gene in itself may lead tocell killing, as with expression of the diphtheria toxin A gene, or theexpression of the gene, may render cells selectively sensitive to thekilling action of certain drugs, e.g., expression of the HSV thymidinekinase gene renders cells sensitive to antiviral compounds includingacyclovir, gancyclovir and FIAU(1-(2-deoxy-2-fluoro-β-D-arabinofuranosil)-5-iodouracil). This cellkilling approach to gene therapy (which may, for instance, be employedin the treatment of cancer) can further be enhanced with use in thevector of a so-called "runaway replication origin" as described furtherherein. However, the use of such a runaway replication system is notlimited to cell killing, and can be employed, for instance, undercircumstances wherein a transient high level of expression is desired(e.g., for a "bolus-type" delivery of a protein by recombinant means).

Moreover, the therapeutic gene can exert its effect at the level of RNA,for instance, by encoding an antisense message or ribozyme, a proteinwhich affects splicing or 3' processing (e.g., polyadenylation), or canencode a protein which acts by affecting the level of expression ofanother gene within the cell (i.e., where gene expression is broadlyconsidered to include all steps from initiation of transcription throughproduction of a processed protein), perhaps, among other things, bymediating an altered rate of mRNA accumulation, an alteration of mRNAtransport, and/or a change in post-transcriptional regulation. Such apassenger gene can encode any other protein, for instance, EBNA-1, andcan be present alone or in addition to further passenger gene(s) locatedbetween the first and second recombining sites.

The passenger gene being transferred can comprise DNA which can be assmall as one repeat unit (i.e., a nucleotide for DNA) and as large asreasonably can be isolated or synthesized, or transferred to a host cellusing the methods of the present invention, and considering packagingconstraints of viral vectors, or upper size limits of plasmid vectors.The passenger gene can constitute or encode coding or non-codingsequences, sense or antisense sequences, including ribozymes, orcatalytic RNA species such as described in the art (Hampel et al.,Nucleic Acids Research, 18, 299-304 (1990); Cech et al., Annual Rev.Biochem., 55, 599-629 (1986)), as well as engineered sequences, orsequences which are not normally present in vivo.

Similarly, the vectors employed in both the parallel and antiparallelrecombination methods may include other genes or coding sequences. Forinstance, a coding sequence or a second coding sequence according to theinvention can comprise the coding sequence of Cre or Flp, the codingsequence of EBNA-1, or any other coding sequence described herein.

In particular, the parallel and antiparallel recombination methods ofthe present invention can be employed to deliver Rep proteins to cells,either as a coding sequence, or a passenger gene. Various mutated Repproteins and variations on the Rep coding sequence such as are known andhave been reported in the literature (see, e.g., McCarty et al., J.Virol., 66, 4050-7 (1992); Kyostio et al., supra) may be employed. Inthis fashion, the methods allow promotion of Rep-mediated recombinationof sequences flanked by one or more adeno-associated virus ITRs (orsequences containing these ITRs that are known to those skilled in theart) into human chromosome 19 or into sequences that have been describedin the art (see, e.g., Linden et al., Proc. Natl. Acad. Sci., 93,7966-7972 (1996)). The methods also can be employed to promoteRep-mediated recombination of sequences flanked by the adeno-associatedvirus ITRs into other genomic regions inasmuch as Rep proteinspreferentially direct recombination into chromosome 19, but can directrecombination to other sites as well. Preferably the viral ITRs havebeen rendered deficient in packaging (e.g., Muzyczka, supra).

Any coding sequence (i.e., a first, second, or other coding sequence) orpassenger gene according to the invention may be followed by sequenceswhich allow production of a polycistronic message. There are a few waysin which such a polycistronic message--which reflects a highly efficientuse of vector space--can be produced. For instance, a ribosome canessentially be recruited to the region of the AUG initiator codon of thesecond message by placing a 300-400 bp fragment from poliovirus inbetween the two coding regions. This allows cap-independent translationbypass, or translation of a non-capped message in a eukaryotic cell.Such a strategy (and hence, such similar sequences allowing use of thestrategy in other species) has also been described in alpha mosaic plantvirus. In a third approach to generation of a polycistronic message, asequence of the cardiovirus is placed in frame between the codingsequences of the polycistronic message. No initiation codon (AUG) isrequired for the coding sequence of the downstream protein. Upontranslation, the proteins are liberated by a mechanism under activeinvestigation. Along the same lines, sequences allowing production ofpolycistronic messages also can be placed after the coding region of thecoding sequence.

Thus, these approaches can be employed to generate a polycistronicmessage from the coding sequence, and coding sequences interveningbetween the first recombining site and the coding sequence. Forinstance, the EBNA-1 coding sequence (or some other coding sequence) canbe the second open reading frame (ORF) in a dicistronic (orpolycistronic) cassette that is activated by recombination. The viralpromoter for EBV that drives expression of EBNA-1 provides only about0.1% of the promoter activity observed with use of the CMV promoter. Ina dicistronic cassette, however, the second gene is expressed at 0.1% ofthe first gene, and 10-fold above background. In this scenario, EBNA-1would be induced by the recombination event. This is especiallydesirable in instances in which EBNA-1 expression during propagation ofviral vectors may potentially act at oriP and destabilize the virus.Alternately, since oriP only replicates once per cell cycle, it can bepossible in some circumstances to create a virus in which EBNA-1 is notregulated, but instead is under the control of a weak constitutivepromoter.

Any suitable combination of recombining sites and recombinase can beutilized with the parallel and antiparallel recombination methods andvectors. Preferably, the first and second recombining sites comprise Loxsites, and the recombinase comprises Cre, or the first and secondrecombining sites comprise Frt sites, and the recombinase comprises Flp.Alternately, the recombinase can be another member of the λ integrasefamily of recombinases (or any other appropriate recombinase), and therecombining site can be the corresponding sequence at which therecombinase acts. The recombinase can be provided to the cell by anysuitable means, e.g., by locating the recombinase coding sequences onthe delivery vector (for instance, as a coding sequence or passengergene), coadministering a second vector which encodes the recombinasegene, or supplying exogenous recombinase.

Preferably the Cre coding sequence is comprised of the coding sequenceof bacteriophage P1 recombinase Cre, or various mutations of thissequence such as described in the art (e.g., Wierzbicki et al., J. Mol.Biol., 195, 785-794 (1987); Abremski et al., J. Mol. Biol., 202, 59-66(1988); Abremski et al., J. Mol. Biol., 184, 211-20 (1988); Abremski etal., Protein Engineering, 5, 87-91 (1992) Hoess et al., Proc. Natl.Acad. Sci., 84, 6840-6844 (1987); Sternberg et al., J. Mol. Biol., 187,197-212 (1986)), or the Cre protein supplied to cells is produced fromone of these sequences by recombinant means. Further mutations of thiscoding sequence which are yet to be isolated also can be employed, solong as variant proteins resulting from such mutations are capable ofeffecting recombination at Lox sites. Furthermore, a nuclearlocalization signal can be appended to the Cre coding sequence toincrease the concentration of Cre protein in the nucleus. Moreover, itis possible to co-supply to a cell vectors encoding complementary Cre orother mutant recombinase proteins, allowing for recombinase activity viaintragenic or intergenic complementation. Assays for Cre function at Loxsites can be carried out as described by Abremski et al. (Abremski etal., J. Biolog. Chem., 259, 1509-1514 (1984)) and Sauer et al. (Sauer etal. (1990), supra).

In terms of Cre/Lox driven recombination, preferably the loxP site isemployed as a Lox site, or various mutations of this sequence areincorporated such as have been described in the literature (see, e.g.,Mack et al., supra; Hoess et al. (1986), supra; Hoess et al.,Biochemistry, 81, 1026-29 (1984); Hoess et al., Gene, 40, 325-329(1985); Abremski et al., J. Biolog. Chem., 261, 391-396 (1986)). Similarmutated sequences of loxP which are yet to be isolated also can beemployed, so long as such sequences are capable of serving asrecombining sites for Cre.

The first, or parallel, recombination method and the second, orantiparallel, recombination method may be better understood withreference to the accompanying drawings as referenced herein,particularly FIGS. 1A-1C through FIGS. 5A-5G which depict variousillustrative embodiments of the parallel and antiparallel recombinationvectors useful in the present inventive parallel and antiparallelrecombination methods, respectively.

a. Parallel Recombination Method

The parallel recombination method of the present invention allows forthe generation of either a miniplasmid or an episome from anextrachromosomal genetic element. Whereas a miniplasmid does notreplicate within a cell (and thus is of use where transient delivery orintegration of a sequence is desired), an episome is capable ofreplicating extragenomically inside a cell. An episome is obtained whenan origin or replication is present in the portion of the recombinationsubstrate which is circularized as a result of the recombination event(as depicted in FIGS. 1A-1C); a miniplasmid is obtained when there is nosuch origin. The parallel recombination method thus has particularutility for viral vectors that are not stably maintained in cells as aconsequence of their inability to integrate as proviruses into the hostgenome, or for viruses such as HSV whose genome is quiescent in thelatent state. This method can be employed to obtain persistent geneexpression by imparting stable maintenance in host cells upon passengergene-carrying vectors which typically are not stably maintained, or arequiescent when latent, and also can be employed to modulate expressionof genes carried by the vectors. Generally, there are threeconsiderations for the delivery of episomes according to this method:(1) an origin of replication, (2) a means of excising the episome fromthe delivery vector, and (3) a method of ensuring segregation of adaughter molecule to each cell at mitosis.

Accordingly, the parallel recombination method of effectingsite-specific recombination in a eukaryotic cell comprises: (a)contacting the cell with a vector comprising an origin of replicationfunctional in mammalian cells located between first and secondrecombining sites located in parallel such that the vector isinternalized by the cell, and (b) providing the cell with asite-specific recombinase that effects recombination between the firstand second recombining sites of the vector.

As shown by way of the preferred vector depicted in FIG. 1A, either alinear virus (i.e., virus sequences indicated by the stippled boxes) ora circular molecule (i.e., plasmid or virus sequences indicated by thestippled line) can be utilized in conjunction with the parallelrecombination method. In accordance with the aforementionedconsiderations for episome delivery, the parallel recombination method,and also, preferably, the antiparallel recombination method, can makeuse of an origin of replication functional in mammalian cells (e.g., anorigin isolated from mammalian cells or a viral replication origin suchas, but not limited to, a replication origin from Ad, HSV, EBV,papillomavirus, vaccinia virus, or polyomavirus. Accordingly, thepresent invention provides a set of vectors that will provide either asingle integrated template, a low or intermediate number of episomes orminiplasmids per cell, or up to thousands of episomes per cell, each ofwhich can comprise various coding sequences, promoters, and/or passengergenes, as described herein.

Desirably, vectors employed in the methods of the present inventionpossess an origin of replication which comprises a sequence of the EBVlatent replicon, oriP, or variations on this sequence that are known andhave been described in the art (e.g., Middleton et al., supra; Reismanet al., supra). This sequence can further comprise variations on theoriP sequence which are yet to be obtained, provided that such mutatedsequences are capable of imparting autonomous replication onextragenomic DNA, as determined using methods known to those skilled inart (e.g., Middleton et al., supra; Reisman et al., supra). Moreover, inthe parallel and antiparallel recombination methods, the origin ofreplication can be any replication origin capable of functioning in anycell, including non-eukaryotic or single-celled eukaryotic cells (e.g.,prokaryotes and yeast) (see, e.g., M. Depamphilis, Ed., DNA Replicationin Eukaryotic Cells (Cold Spring Harbor Press, NY, 1996)).

Notably, oriP serves a different biological function than do manyorigins of DNA replication that have been characterized from viruses.Namely, oriP permits controlled replication of plasmid DNA such thatcell survival is not impaired. In contrast, other replication origins,termed "runaway replication origins", permit an exponential rate of DNAreplication during the cell cycle, and ultimately can lead to celldeath, depending on what other gene are present on the vector. Suchrunaway replication origins include but are not limited to thereplicative origins of SV40, BK, polyomavirus, Ad, and HSV-1. Theserunaway replication origins also preferably can be employed in themethods and vectors of the present invention, and can be used forapplications other than cell killing. Of course, other cofactors mayneed to be co-supplied to the cell for the origins to function properlyin the cell.

In particular, with use of a polyomavirus runaway replication origin(e.g., as described by Muller, Mol. Cell. Biol., 8, 5000-5015 (1988),and in GenBank Database Accession Number J02288 or PLY 2CG), viral largeT antigen is required to initiate viral DNA replication. This proteinacts by binding within the region of the replication origin, unwindingthe DNA in this vicinity, and allowing other cellular enzymes (such asDNA polymerases) to act. Thus, with use of such a polyomavirusreplication origin, at least viral T antigen preferably also is providedto the cell, for instance, as either a coding sequence (e.g., a first,second, or other coding sequence), or a passenger gene (i.e., in cis orin trans). Viral T antigen also can be provided exogenously. In somecases, it may be desirable to provide further permissive factors, e.g.,to optimize viral replication. Desirably, the polyomavirus originemployed is a mouse polyomavirus origin. However, also preferably, theorigin is a human, or other, polyomavirus origin. Sequences encodingthese origins, as well as variations of these sequences, are known inthe art (Muller, supra).

Similarly, an origin that permits a multicopy number of episomes percell to be obtained can be employed. For instance, a papillomavirusorigin (e.g., described in GenBank Database Accession Number PAPABPV1,with the BPV1 complete genome being described in GenBank DatabaseAccession Number X02346, and the BPV4 early coding sequence beingdescribed in GenBank Database Accession Number D00146) can be used inthe vectors of the invention. Papillomavirus genomes are maintained asmulticopy plasmids in transformed cells. When such a papillomavirusorigin is employed, desirably the viral E1 protein also is provided tothe cell (e.g., as a coding sequence or passenger gene located in cis orin trans, or provided exogenously). In some cases, it also may bedesirable to similarly supply the cell with a source of viral E2 proteinto optimize replication. The papillomavirus origin and E1 and E2proteins are known to those skilled in the art, and these sequences, orvariations of these sequences, can be used (see, e.g., Pirsoo et al.,EMBO J., 15, 1-11 (1996), Grossel et al., Virology, 217, 301-310(1996)). In particular, preferably the papillomavirus origin is that ofa bovine. Alternately, preferably the papillomavirus is that of a human,but can be of any other source of the virus.

While these, and other, origins of replication that similarly impartability on a circular DNA element to replicate autonomously within thehost cell genome can be employed instead of oriP, when the oriP originis employed, EBNA-1 also can be supplied to the cell. As for the othercell replication proteins, EBNA-1 protein can be provided exogenously,or the EBNA-1 coding sequence can be provided in cis or in trans tooriP, in the same manner as described for delivery of recombinase to thecell. Even though plasmids possessing oriP in the absence of EBNA-1protein can be stably maintained in cells which have been infected withEBV (Lupton et al., supra; Teshigawara et al., Nuc. Acids Res., 20, 2607(1992)), the coding sequences for EBNA-1 together with oriP constitutethe minimal requisite components for stable maintenance of a EBV-derivedcircular plasmid in cultured cells in the absence of EBV infection.Moreover, as described further herein, the parallel recombination methodmay be employed for delivery of Rep proteins (as well as other proteins)to a cell wherein an origin of replication is not utilized.

The excision of the episome or miniplasmid from the parallelrecombination vector and its subsequent circularization is accomplishedthrough application of a recombination system consisting of first andsecond recombining sites (FRS and SRS in FIG. 1A) within the vector, anda site-specific recombinase which acts at the first and secondrecombining sites to effect recombination between the first and secondrecombining sites of the vector. The presence of two recombining siteslocated in parallel on a single DNA molecule leads to excision by theappropriate recombinase of the intervening sequences so as to form aclosed circular molecule. Partitioning of the episome between daughtercells can be accomplished through application of EBNA-1, which appearsto act by binding to the host cell genome and oriP (or other origin ofreplication) and essentially pulling the episome into the nascent cell,or a protein having a similar function.

Accordingly, by encoding oriP, or another origin of replicationfunctional in mammalian cells, between two parallel recombining sites inthe vector, an episome will be generated by recombination uponappropriate application of the recombinase to the cell. Namely, uponproviding the cell which has internalized the parallel recombinationvector with such a site-specific recombinase, the parallel recombinationvector (as exemplified by FIG. 1A) is recombined between the first andsecond recombining sites so as to form an episome (as exemplified inFIG. 1C). A miniplasmid similarly can be generated wherein there is noorigin of replication present in between the parallel recombining sites.When a viral vector is employed as an episome or miniplasmid deliveryvehicle, the second product of the recombination event (as exemplifiedin FIG. 1B) is a truncated virus, and when a plasmid is employed, atruncated plasmid, or miniplasmid, will similarly result.

Preferably, the vector further comprises the coding sequence for therecombinase, particularly the coding sequence for Cre. The vector alsopreferably can comprise one or more additional coding sequences, such asa first, second, and/or third coding sequence, and/or an additionalcoding sequence comprising a promoter, such as a passenger gene. Thus,in the parallel recombination method, the vector preferably furthercomprises a passenger gene. Optimally the passenger gene is locatedbetween the first and second recombining sites, as depicted in theparallel recombination vector of FIG. 1A, which results in an episomewhich carries the passenger gene (PG), as depicted in FIG. 1C.

Similarly, preferably the vector further comprises a coding sequence.Such a coding sequence can be any suitable coding sequence, optimally anEBNA-1 or Rep coding sequence, or various mutations of these sequenceswhich are known and have been described in the art (see, e.g., Yates etal. (1985), supra; Lupton et al., supra, Middleton et al., J. Virol.,66, 1795-1798 (1992); Levitskaya et al., supra). When replicationorigins other than oriP are employed, other viral replication proteinssimilarly can be provided as a coding sequence (e.g., large T antigen,E1, or E1 and E2). Desirably the coding sequence is located between thefirst and second recombining sites containing the replication origin,resulting in an episome carrying the coding sequence.

Along these lines, the parallel recombination method makes it possibleto up- or down-regulate one coding sequence, or to simultaneouslyup-regulate one coding sequence and down-regulate another, through therecombination event. For instance, as depicted in FIG. 1A, a firstcoding sequence (FCS) can be located between the first and secondrecombining sites (FRS and SRS) and oriented to be transcribed in thedirection from the first recombining site (FRS) to the secondrecombining site (SRS). On the same DNA molecule, a promoter (P) can belocated between the FCS and SRS, such that P is capable of transcribinga coding sequence downstream of SRS. In such a situation, prior torecombination, the FCS will not be transcribed into nascent mRNA.However, following recombination, the FCS will be transcribed, as aconsequence of the recombination event placing the FCS under the controlof P.

The parallel recombination method also makes it possible todown-regulate transcription of one coding sequence simultaneous withup-regulation of another coding sequence, through the recombinationevent. Namely, if a second coding sequence (SCS) is located immediatelydownstream of P, and is operably linked to P such that P is capable ofcontrolling the expression of the SCS, then prior to recombination, theSCS will be transcribed. After recombination, P, which previouslycommanded transcription of the SCS, will be placed upstream of the FCS,and the FCS will be transcribed, whereas the SCS will not betranscribed. It is not necessary, however, that the promoter control theexpression of any downstream sequence prior to recombination. Similarly,it is not necessary that the promoter control expression of anothercoding sequence after recombination. This method can also allowindependent down-regulation of a single coding sequence, e.g., byincorporating SCS in the vector of FIG. 1A in the absence of FCS.

Thus, in one embodiment of the parallel recombination method which canbe employed to up-regulate a coding sequence, the method is preferablycarried out wherein: (a) the coding sequence is located in the regionbetween the first and second recombining sites which comprises theorigin and adjoins the first recombining site such that the codingsequence is oriented to be transcribed in the direction from the firstrecombining site to the second recombining site proceeding through thecoding sequence, (b) the coding sequence is not operably linked to anypromoter, and (c) a promoter is located in the region between the firstand second recombining sites which comprises the origin and adjoins saidsecond recombining site such that the promoter is oriented to directtranscription in the direction from the first recombining site to thesecond recombining site proceeding through the promoter. Optimally thereare no coding sequences, promoters, or transcription termination siteslocated between the first recombining site and the coding sequence.

Preferably, the vector employed to up-regulate expression of one codingsequence further comprises a second coding sequence located in a regionother than the region between the first and second recombining siteswhich comprises said origin and adjoining the second recombining site,and which is operably linked to the promoter. This method allows forsimultaneous up- and down-regulation of separate coding sequences.Optimally there are no coding sequences, promoters, or transcriptiontermination sites located between the promoter and the second codingsequence.

In another embodiment of the parallel recombination method which can beemployed to independently down-regulate a coding sequence in the absenceof up-regulation of another sequence, the method is preferably carriedout wherein: (a) a promoter is located in the region between the firstand second recombining sites which comprises the origin and adjoins saidsecond recombining site such that the promoter is oriented to directtranscription in the direction from the first recombining site to thesecond recombining site proceeding through the promoter, and (b) thecoding sequence is located in a region other than the region between thefirst and second recombining sites which comprises the origin andadjoins the second recombining site such that the coding sequence isoperably linked to the promoter.

These further methods also preferably can be employed when the episomedelivery vector additionally carries a passenger gene. Moreover, tooptimize production of protein encoded by the coding sequence followingrecombination, splice donor and acceptor sites can be included in thevector employed in the parallel recombination method.

According to this invention, any promoter (e.g., a promoter which islinked with a coding sequence as a consequence of recombination, as wellas a promoter present as part of a passenger gene) whether isolated fromnature, or produced by recombinant DNA or synthetic techniques, may beused to provide for gene transcription, so long as the promoterpreferably is capable of directing transcription in a eukaryotic(desirably mammalian) cell.

The DNA sequences appropriate for expression in eukaryotic cells (i.e.,"eukaryotic promoters") differ from those appropriate for expression inprokaryotic cells. Generally, eukaryotic promoters and accompanyinggenetic signals are not recognized in or do not function in prokaryoticsystems and, prokaryotic promoters are not recognized in or do notfunction in eukaryotic cells.

A comparison of promoter sequences that function in eukaryotes hasrevealed conserved sequence elements. Generally, eukaryotic promoterstranscribed by RNA polymerase II are typified by a "TATA box" centeredaround position -25 which appears to be essential for accuratelypositioning the start of transcription. The TATA box directs RNApolymerase to begin transcribing approximately 30 bp downstream inmammalian systems. The TATA box often functions in conjunction with atleast two other upstream sequences located about 40 bp and 110 bpupstream of the start of transcription. Typically, a so-called "CCAATbox" serves as one of the two upstream sequences, and the other often isa GC-rich segment (e.g., a "GC box" comprised, for instance, of thesequence GGGCGG, or the sequences GCCACACCC and ATGCAAAT). The CCAAThomology can reside on different strands of the DNA. The upstreampromoter element may also be a specialized signal such as those whichhave been described in the art and which seem to characterize a certainsubset of genes.

To initiate transcription, the TATA box and the upstream sequences areeach recognized by regulatory proteins which bind to these sites, andactivate transcription by enabling RNA polymerase II to bind the DNAsegment and properly initiate transcription. Base changes within theTATA box and the upstream sequences can substantially lowertranscription rates (e.g., Myers et al., Science, 229, 242-7 (1985);McKnight et al., Science, 217, 316-324 (1982)). The position andorientation of these elements relative to one another, and to the startsite, are important for the efficient transcription of some, but notall, coding sequences. For instance, some promoters function well in theabsence of any TATA box. Similarly, the necessity of these and othersequences for promoters recognized by RNA polymerase I or III, or otherRNA polymerases, can differ.

Accordingly, promoter regions vary in length and sequence, and canfurther encompass one or more DNA-binding sites for sequence-specificDNA binding proteins, and/or an enhancer or silencer. The presentinvention preferentially employs a CMV promoter, or a P5 promoter as apromoter for regulating a gene or coding sequence of interest (e.g., a"first coding sequence" or "second coding sequence" as described furtherherein). Such promoters, as well as mutations thereof, are known andhave been described in the art (see, e.g., Hennighausen et al., EMBO J.,5, 1367-1371 (1986); Lehner et al., J. Clin. Microbiol., 29, 2494-2502(1991); Lang et al., Nucleic Acids Res., 20, 3287-95 (1992); Srivastavaet al., J. Virol., 45, 555-564 (1983); Green et al., J. Virol., 36,79-92 (1980); Kyostio et al., supra). Other promoters, however, can alsobe employed, such as the Ad2 or Ad5 major late promoter and tripartiteleader, the Rous sarcoma virus (RSV) long terminal repeat, and otherconstitutive promoters such as have been described in the literature.For instance, the herpes thymidine kinase promoter (Wagner et al., Proc.Natl. Acad. Sci., 78, 144-145 (1981)), the regulatory sequences of themetallothionine gene (Brinster et al., Nature, 296, 39-42 (1982))promoter elements from yeast or other fungi such as the Gal 4 promoter,the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter,and the alkaline phosphatase promoter can be employed. Similarly,promoters isolated from the genome of mammalian cells or from virusesthat grow in these cells (e.g., Ad, SV40, CMV, and the like) can beused.

Instead of using a constitutive promoter, the promoter also preferablycan be up- and/or down-regulated in response to appropriate signals. Forinstance, an inducible promoter, such as the IL-8 promoter, which isresponsive to TNF or another cytokine can be employed. Other examples ofsuitable inducible promoter systems include, but are not limited to, themetallothionine inducible promoter system, the bacterial lac expressionsystem, and the T7 polymerase system. Further, promoters that areselectively activated at different developmental stages (e.g., globingenes are differentially transcribed in embryos and adults) can beemployed. In particular, a promoter that can be regulated by exogenousfactors such as tetracycline, or synthetic hormone such as RU-486 can beemployed. These promoters (and accompanying regulatory factors thatsimilarly can be provided to the cell) have all been described in theart (see, e.g., Delort et al., Human Gene Therapy 7, 809-820 (1996);Clontech, CLONTECHniques, "Tet-Off™ and Tet-On™ Gene Expression Systemsand Cell Lines", Volume XI, No. 3, 2-5 (July 1996)).

Another option is to use a tissue-specific promoter (i.e., a promoterthat is preferentially activated in a given tissue and results inexpression of a gene product in the tissue where activated), such as thehepatocyte-specific promoter for albumin or a₁ -antitrypsin (Frain etal., Mol. Cell. Biol. 10: 991-999 (1990); Ciliberto et al., Cell 41:531-540 (1985); Pinkert et al., Genes and Devel., 1, 268-276 (1987);Kelsey et al, Genes and Devel., 1, 161-171 (1987)), the elastase I genecontrol region which is active in pancreatic acinar cells (e.g., Swiftet al., Cell, 38, 639-646 (1984); MacDonald, Hepatology, 7, 425-515(1987)), the insulin gene control region which is active in pancreaticbeta cells (Hanahan, Nature, 315, 115-122 (1985)), the mouse mammarytumor virus control region which is active in testicular, breast,lymphoid and mast cells (Leder et al., Cell, 45, 485-495 (1986)), themyelin basic protein gene control region which is active inoligodendrocyte cells in the brain (Readhead et al., Cell, 48, 703-712(1987)), and the gonadotropic releasing hormone gene control regionwhich is active in the hypothalamus (Mason et al., Science, 234,1372-1378 (1986)). Similarly, a tumor-specific promoter, such as thecarcinoembryonic antigen for colon carcinoma (Schrewe et al., Mol. CellBiol. 10: 2738-2748 (1990)) can be used in the vector. According to theinvention, any promoter can be altered by mutagenesis, so long as it hasthe desired binding capability and promoter strength.

Additionally, it is now known that inducible activity can be obtained byfusing the C-terminal ligand binding domain of a hormone receptor ontothe coding sequence of a particular protein (see, e.g., Kellendock etal., Nucleic Acids Res., 24, 1404-1411 (1996); Metzger et al., Proc.Natl. Acad. Sci., 92, 6991-6995 (1995)). This approach is advantageousin that when employed to create a recombinase fusion, it renders therecombination event inducible, and under the control of an exogenoussubstance that binds the steroid receptor. However, the method alsopreferably is employed with any coding sequence (e.g., a first andsecond coding sequence) according to the invention and/or any passengergene. In particular, the method preferably is employed with use of aligand binding domain (e.g., a hormone binding domain) of a hormonereceptor that is capable of binding exogenous, but notnaturally-occurring, steroid. For instance, the ligand binding domain ofa mutant hormone progesterone receptor (hPR891) that responds to a lowlevel of synthetic steroid such as RU-486, but not to endogenoussteroid, can be employed (Kellendock et al., supra). Alternately, ligandbinding domains from estrogen, glucocorticoid, and androgen receptorscan be fused to the recombinase, or other coding sequence. Other similarapproaches also have been described in the art and can be employedherein to render the activity of the recombinase or other proteinencoded by a coding sequence according to the inventionligand-dependent, either through creation of translational fusionsresulting in a chimeric protein, or through transcriptional fusionsplacing the gene under the control of an exogenous substance, aspreviously described. Moreover, unique proteins such as rapamycin allowthe linkage of proteins that normally ignore each other, adding yetanother level of control to the systems described herein (see, e.g.,Balter et al., Science, 273, 183 (1996); Choi et al., Science, 273,239-242 (1996)).

Furthermore, in some instance, the origins employed for autonomousreplication of the vectors may also comprise sequences that function aspromoters (e.g., due to the close intertwinement of DNA replication andtranscription). For instance, the polyomavirus origin contains aconstitutive promoter that can be employed, for instance to driveexpression of a passenger gene. Similarly, the bovine papillomavirusorigin of replication comprises a promoter that is induced by thepapillomavirus E2 protein. This origin potentially can be used as ameans of a gene within a vector according to the invention. Similarly,the EBV origin contains repeats that act as an enhancer in the presenceof EBNA-1. These repeats can be employed, for instance, to up-regulateexpression of a gene upon recombination-mediated up-regulation ofEBNA-1.

The ordinary skilled artisan is aware that different genetic signals andprocessing events control levels of nucleic acids and proteins/peptidesin a cell, such as, for instance, transcription, mRNA translation, andpost-transcriptional processing. Transcription of DNA into RNA requiresa functional promoter. The amount of transcription is regulated by theefficiency with which RNA polymerase can recognize, initiate, andterminate transcription at specific signals. These steps, as well aselongation of the nascent mRNA and other steps, are all subject to beingaffected by various other components also present in the cell, e.g., byother proteins which can be part of the transcription process, byconcentrations of ribonucleotides present in the cell, and the like.

Protein expression is also dependent on the level of RNA transcriptionwhich is regulated by DNA signals, and the levels of DNA template.Similarly, translation of mRNA requires, at the very least, aninitiation codon (preferably an AUG initiation codon) which is usuallylocated within 10 to 100 nucleotides of the 5' end of the message.Sequences flanking the AUG initiator codon have been shown to influenceits recognition by eukaryotic ribosomes, with conformity to a perfectKozak consensus sequence resulting in optimal translation (see, e.g.,Kozak, J. Molec. Biol., 196, 947-950 (1987)). Also, successfulexpression of a foreign nucleic acid sequence in a cell can requirepost-translational modification of a resultant protein/peptide. Thus,production of a recombinant protein or peptide can be affected by theefficiency with which DNA is transcribed into mRNA, the efficiency withwhich mRNA is translated into protein, and the ability of the cell tocarry out post-translational modification. These are all factors ofwhich the ordinary skilled artisan is aware and is capable ofmanipulating using standard means to achieve the desired end result.

Along these lines, to optimize protein production followingrecombination, preferably the vector employed for simultaneous up- anddown-regulation of separate coding sequences further comprises: (a) asplice acceptor site located between the first recombining site and thecoding sequence in the region between the first and second recombiningsites which comprises the origin, and (b) a splice donor site locatedbetween the promoter and the second recombining site in the regionbetween the first and second recombining sites which comprises theorigin, and (c) a splice acceptor site located between the secondrecombining site and the second coding sequence in a region other thanthat including the region between the first and second recombining siteswhich comprises the origin.

The ordinary skilled artisan also is aware that the immune response tocertain immunogenic proteins can result in a decreased level of theprotein in vivo. The Cre protein is an example of such a protein againstwhich an immune response could be generated. In comparison, the EBVEBNA-1 protein efficiently escapes detection by the immune system.Accordingly, the present invention also provides a method of renderingCre or other similar immunogenic proteins employed in the context of theinvention invisible to the immune system. This method comprises placingcopies of the glycine-alanine repeat of the EBNA-1 protein (orconservative variations thereof) at either the C- or N-terminus of theprotein. Such placement preferably is done at the level of DNA in acoding sequence (e.g., of a first or second coding sequence, othercoding sequence, or passenger gene). The glycine-alanine repeats areknown in the art (see, e.g., Yates et al., supra; Lupton et al., supra;Levitskaya et al., supra). Preferably this is done by incorporating thenucleic acid sequence of the glycine-alanine repeats (or variants) ateither the 5' or the 3' end of the coding sequence of interest. Inparticular, this approach also can be employed with use of a ligandbinding domain, as previously described. As illustrated in FIGS. 2A-2F,the glycine-alanine repeats (Gly Ala) can be employed in a variety oflocations vis-a-vis the coding sequence (CS) and ligand binding domain(HBD), if present.

The parallel recombination method can be employed to provide temporalregulation of genes and coding sequences. For instance, the first codingsequence can be an EBNA-1 or Rep coding sequence, as previouslydescribed. Also, the second coding sequence can comprise the codingsequence for Cre or Flp, as previously described. More generally,however, either the first or second (or other) coding sequence cancomprise either the EBNA-1, Rep, Flp, Cre, large T antigen, E1, or E2coding sequences.

The regulation effected due to the recombination event can be alsoemployed to provide temporal expression of the recombinase codingsequence. Namely, the coding sequence for the recombinase can be locatedin the position of the SCS in FIG. 1A. Following excision of theepisome, the recombinase will no longer be produced in the host cell.This approach is advantageous in that it is preferable that recombinaseactivity not occur following episome generation. This reduces thepossibility of reintegration of the episome, or multimerization due tointermolecular recombination.

Along the same lines, the coding sequence for EBNA-1 can be located inthe position of the FCS in FIG. 1A. This allows regulation of expressionof the EBNA-1 coding sequence which is advantageous sinceEBNA-1-mediated replication of oriP in cis during viral production(i.e., when a virus is employed as the episome delivery vector) may, insome cases, lead to undesirable genome instability and a reduced yield.If the EBNA-1 coding sequence is provided as the FCS, then itsexpression will only occur after excision of the episome, when it ismost needed.

Production of EBNA-1, or any protein encoded by a coding sequence whosetranscription is commanded by P (i.e., FCS or SCS), can be maximized byplacing SRS inside of an intron. This precludes the possibility ofinitiation at any fortuitous initiation codons present within SRS, andsimilarly precludes the formation of any fusion proteins. This can beaccomplished, as illustrated in FIG. 1A, by locating splice acceptorsites (SAs) between FRS and FCS, and between SRS and SCS, and by alsolocating a splice donor site (SD) between P and SRS. Production ofprotein encoded by the FCS, SCS, or TG similarly can be maximized bypositioning polyadenylation sites immediately downstream of the codingregions for these sequences.

The approach employed for controlling expression of EBNA-1 similarly canbe employed as a means of controlling the transcription and subsequenttranslation of the Rep proteins from AAV. Temporal expression andexpression levels of the Rep gene desirably is tightly regulated toavoid two potential problems. First, similar to EBNA-1, the presence ofRep proteins in the host cell during virus production could cause viralinstability and decreased yields when a virus is employed as the episomedelivery vector. Second, overexpression of the Rep gene is cytotoxic tothe cell and could elicit an immune response. These potential problemscan be avoided by cloning a Rep coding sequence into the position of theFCS in FIG. 1A. This will insure an absence of Rep gene expressionduring viral production. Upon infection and delivery of recombinase tocells containing the Rep-encoding vector (e.g., either by supplying thecoding sequence for recombinase on the same vector, possibly as the SCS,or on a co-administered vector), recombination will occur, bringing Pupstream of the Rep coding sequence, and placing the Rep coding sequenceunder the control of P. The P5 promoter, which is the natural promoterfor the Rep gene, can be employed as P, since this promoter shouldcommand appropriate levels of Rep gene expression to allow Rep-mediatedgenomic integration without cytotoxic effects or eliciting an immuneresponse.

Sequences flanked by one or more AAV ITRs are preferentially integratedinto human chromosome 19 when Rep proteins are present. Thus in oneembodiment of the method of the present invention, the coding sequenceis a Rep coding sequence. Preferably the vector further comprises one ormore (e.g., first and second) adeno-associated virus ITRs. In the methodof the present invention, a variety of locations of the AAV ITRs can beemployed inasmuch as, theoretically, the ITRs are location-independent.Accordingly, in this method, preferably one or more AAV ITRs are locatedin the region between the first and second recombining sites whichcomprises the origin. One or more ITRs, however, also can be located ina region other than the region between the first and second recombiningsites which comprises the origin. The positioning of one or more AAVITRs immediately flanking the passenger gene will allow integration ofthis gene into another genetic location. It is also possible to locateone or more AAV ITRs so that, upon Rep-directed recombination, Repproteins are inactivated. For instance, one or more AAV ITRs can beplaced between the FCS encoding Rep and a downstream polyadenylationsite. In this case, Rep-mediated recombination of the episomal sequencesinto another genetic site would sever the Rep coding sequence from thepolyadenylation site, thus abrogating Rep activity since transcripts arerapidly degraded in eukaryotic cells unless polyadenylated. Likewise,other genes similarly can be regulated, activated, or inactivated usingthe parallel recombination method of the present invention.

Alternately, Rep proteins can be delivered to the cell by providing themencoded on a miniplasmid generated using the parallel recombinationmethod. This approach, as previously described, involves the generationof a miniplasmid on which Rep or another protein coding sequence iscontained through the use of a vector comprising the sequence ofinterest located between two directly repeated recombining sites,wherein there is no origin of replication that similarly is locatedbetween the recombining sites.

Accordingly, the method of providing Rep proteins to a cell comprises:(a) contacting the cell with a vector comprising a Rep coding sequencelocated between first and second recombining sites located in parallelsuch that the vector is internalized by the cell, and (b) providing thecell with a site-specific recombinase that effects recombination betweenthe first and second recombining sites of the vector. Optimally the Repcoding sequence is operably linked to a promoter within the vector, oris operably linked to a promoter within the vector as a consequence ofthe recombination event.

Preferably this method is carried out wherein: (a) the Rep codingsequence adjoins the first recombining site such that the codingsequence is oriented to be transcribed in the direction from the firstrecombining site to the second recombining site proceeding through thecoding sequence, (b) the coding sequence is not operably linked to anypromoter, and (c) a promoter is located in the region between the firstand second recombining sites which comprises the coding sequence andadjoins the second recombining site such that the promoter is orientedto direct transcription in the direction from the first recombining siteto the second recombining site proceeding through the coding sequence.

Also, preferably the vector employed for the delivery of Rep proteinsfurther comprises first and second adeno-associated virus ITRs.Optimally one or more ITRs are located in the region between the firstand second recombining sites which comprises the coding sequence.Alternately, one or more ITRs are located in a region other than theregion between the first and second recombining sites which comprisesthe origin.

Some of these embodiments of the parallel recombination method aredepicted in FIGS. 3A-3X. In particular, FIG. 3A depicts the method ofthe invention wherein a vector comprising parallel recombination sites(FRS and SRS), and an origin of replication located between these sitesis provided to a cell. Recombinase also preferably is provided to thecell, either in cis or in trans. The vector (either linear or circular)employed in this method also preferably comprises a coding sequence (CS)that can be located anywhere on the vector (FIG. 3B), in theori-containing region between the first and second recombining sites(FIG. 3C), or in a region other than the region between the first andsecond recombining sites (FIG. 3D). The vector further preferably cancomprise one or more ITRs located anywhere on the vector (FIG. 3E),and/or can comprise one or more passenger genes (PG; FIG. 3F).

When a promoter is located in the region between the first and secondrecombining sites containing the origin, the parallel recombinationmethod can be employed to down-regulate a coding sequence (FIG. 3G),up-regulate a coding sequence (FIG. 3H), or simultantaneouslyup-regulate a first coding sequence (CS) and down-regulate a secondcoding sequence (SCS; FIG. 3I) by the recombination event. One or moresplice donor (SD) and/or splice acceptor (SA) sites can be incorporatedinto the vector to optimize protein production (FIG. 3J).

Regulation of gene expression effected by the recombination eventsimilarly can be accomplished with use of a vector having a promoterlocated in a region other than the ori-containing region between thefirst and second recombining sites. Such a vector can be employed toup-regulate a coding sequence (FIG. 3K), or simultantaneouslyup-regulate a first coding sequence and down-regulate a second codingsequence (FIG. 3L) by the recombination event. The parallelrecombination method also can be employed with use of a second promoter(SP) to simultaneously up-regulate a first and second coding sequence bythe recombination event (FIG. 3M). Furthermore, a third coding sequencecan be inserted in the vector, between the first promoter and firstrecombining site (FIG. 3N). In particular, when the third codingsequence is oriented in the same direction as the first promoter (FIG.3N), a dicistronic message can be created by the recombination event(e.g., when an internal ribosome entry site also is appropriatelylocated in the vector). When the third coding sequence is oriented inthe opposite direction as the first promoter (FIG. 3N), the third codingsequence is down-regulated by the recombination event while the firstcoding sequence is up-regulated (e.g., optimally when splice donor andacceptor sites are appropriately located in the vector).

In particular, the parallel recombination method provides a convenientmethod for providing Rep protein to a cell, for instance, wherein theRep gene is initially silent and is up-regulated by the recombinationevent (FIG. 30). In this, as in other embodiments, the vector furthercan incorporate one or more ITRs located anywhere on the vector (FIG.30), in the ori-containing region between recombining sites (FIG. 3P),or outside of this ori-containing region (FIG. 3Q).

The parallel recombination method also can be carried out using a vectorthat does not comprise an origin of replication between the first andsecond recombining sites (FIG. 3R). Optionally, this method also can becarried out to deliver Rep to a cell, either as a passenger gene orcoding sequence that is present on the minicircle formed by therecombination event (FIG. 3S). Such a vector further can compriseanother coding sequence that is up-regulated by the recombination event(FIG. 3T). As with use of an ori-containing vector, the vector lackingthe replication origin can be employed to up-regulate a coding sequence(FIG. 3U), simultaneously up-regulate a first coding sequence anddown-regulate a second coding sequence (FIG. 3V), and up-regulate both afirst and second coding sequence (FIG. 3W) by the recombination event.This embodiment also can be applied with use of a third coding sequence(FIG. 3X) as previously described. According to the invention, any ofthe coding sequences (i.e., first, second, or third) and/or thepassenger gene can encode EBNA-1, Rep, Flp, Cre, large T antigen, E1,and E2 coding sequences. Further variations of this method will beapparent to one of skill in the art.

b. Antiparallel Recombination Method

The antiparallel recombination method is similar to that of the parallelrecombination method in that it relies on site-specific recombinationeffected by a suitable recombination system, such as, preferably, thephage P1 Cre/Lox system, the yeast Flp/Frt system, or other appropriaterecombination system. In contrast to the parallel recombination method,however, the antiparallel recombination method involves a vector withthe first and second recombining sites located in an antiparallelorientation on the same DNA molecule. This alternate placement of therecombining sites leads to inversion of the intervening sequences uponapplication of the recombinase, instead of episome excision. Theantiparallel recombination method can be employed to modulate geneexpression, for instance, by providing for the up- or down-regulation ofone coding sequence, or simultaneous up-regulation of one codingsequence and down-regulation of another coding sequence through therecombination event.

Along these lines, the antiparallel recombination method of effectingsite-specific recombination in a cell comprises: (a) contacting the cellwith a vector such that the vector is internalized by the cell, whereinthe vector comprises (i) a promoter located between first and secondrecombining sites in antiparallel orientations and adjoining the secondrecombining site such that the promoter is oriented to directtranscription in the direction from the first recombining site to thesecond recombining site proceeding through the promoter, and (ii) acoding sequence located in a region other than the region between thefirst and second recombining sites which comprises the promoter andadjoining the first recombining site such that the coding sequence isoriented to be transcribed in the opposite direction from the directionof the first recombining site to the second recombining site proceedingthrough the promoter, wherein the coding sequence is not operably linkedto any promoter; and (b) providing the cell with a site-specificrecombinase that effects recombination between the first and secondrecombining sites of the vector. Preferably there are no codingsequences, promoters, or transcription termination sites located betweenthe first recombining site and the coding sequence.

This method preferably further comprises a second coding sequencelocated in a region other than the region between the first and secondrecombining sites which comprises the promoter and adjoining the secondrecombining site, and which is operably linked to the promoter.Optimally there are no coding sequences, promoters, or transcriptiontermination sites located between the promoter and the second codingsequence.

Moreover the antiparallel recombination method also can be employed todown-regulate a single coding sequence in the absence of up-regulationof another coding sequence. This method comprises: (a) contacting thecell with a vector such that the vector is internalized by the cell,wherein the vector comprises: (i) a promoter located between first andsecond recombining sites in antiparallel orientations and adjoining thesecond recombining site such that the promoter is oriented to directtranscription in the direction from the first recombining site to thesecond recombining site proceeding through the promoter, and (ii) acoding sequence located in a region other than the region between thefirst and second recombining sites which comprises the promoter andadjoining the second recombining site such that the coding sequence isoperably linked to the promoter; and (b) providing the cell with asite-specific recombinase that effects recombination between the firstand second recombining sites of the vector. Optimally there are nocoding sequences, promoters, or transcription termination sitesintervening between the promoter and the coding sequence followingrecombination.

Also, preferably, any of the vectors employed for up- and/ordown-regulation of a coding sequence may further comprise a passengergene, and/or the coding sequence of either the Rep, EBNA-1, Cre or Flpgenes, as previously described. As for the parallel recombination methodany coding sequence (e.g., first, second, or other coding sequence)and/or passenger gene can comprise the EBNA-1, Rep, Flp, Cre, large Tantigen, E1, or E2 coding sequences.

Moreover, to optimize protein production following recombination,preferably the vector employed for simultaneous up- and down-regulationof separate coding sequences further comprises: (a) a splice acceptorsite located between the first recombining site and the coding sequencein a region other than that including the region between the first andsecond recombining sites which comprises the promoter, (b) a splicedonor site located between the promoter and the second recombining sitein the region between the first and second recombining sites whichcomprises the promoter, and (c) a splice acceptor site located betweenthe second recombining site and the second coding sequence in a regionother than that including the region between the first and secondrecombining sites which comprises the promoter.

FIG. 4 depicts a preferred vector for use in conjunction with theantiparallel recombination method. As illustrated in FIG. 4, theantiparallel recombination vector comprises a first coding sequence(FCS) adjoining the first recombining site (FRS) such that the FCS isoriented to be transcribed in the opposite direction from the firstrecombining site (FRS) to the second recombining site (SRS). The FCS isnot transcribed, since it is not operably linked to any upstreampromoter. A second coding sequence (SCS) is located adjoining the secondrecombining site (SRS), and is oriented to be transcribed in thedirection from the first recombining site (FRS) to the secondrecombining site (SRS).

The SCS need not necessarily be present, for instance, in the case wherethe expression of one coding sequence is up-regulated, but anothercoding sequence is not down-regulated by the recombination event.However, when the SCS is present, it is transcribed, at least initially,since it is operably linked to a promoter (P) located between the firstand second recombining sites (FRS and SRS). Similarly, the FCS need notnecessarily be present, for instance, in the case where the expressionof one coding sequence is down-regulated, but another coding sequence isnot up-regulated by the recombination event. However, when the FCS ispresent, it is transcribed following recombination, which places Pupstream of the FCS.

Thus, prior to the application of recombinase to a cell containing thisvector, the SCS is transcribed, whereas the FCS is not transcribed.Following application of recombinase, the recombination event places Pupstream of the FCS, and the FCS is transcribed whereas the SCS is nottranscribed. Preferably there are no coding sequences, further promotersor transcription termination sites located between the FCS and the FRS,nor between the SCS and the SRS.

Moreover, to maximize gene expression and protein production, spliceacceptor sites (SAs) can be placed between the FCS and the FRS, andbetween the SCS and the SRS. Moreover, a splice donor site (SD) can beplaced between P and the SRS. Similarly, polyadenylation sites can beinserted following the coding regions of the FCS and SCS sequences.

In such a recombination method, preferably the SCS can encode arecombinase coding sequence, and preferably the FCS can encode a Repcoding sequence, or an EBNA-1 coding sequence. The antiparallelrecombination vector illustrated in FIG. 4, and variations thereof,similarly can be employed for other genes, for instance for genesrequiring proper temporal expression, such as developmentally-regulatedgenes, or for genes whose expression must be stringently controlled,such as those encoding toxic or deleterious proteins.

Moreover, in accordance with the antiparallel recombination method, itis also possible to up- and/or down-regulate a coding sequence (e.g.,such as a coding sequence for Rep, EBNA-1 or recombinase) by placing thecoding sequence in between the recombining sites, and placing one ormore promoters outside the recombining sites. In one embodiment allowingfor up-regulation, this method comprises: (a) contacting the cell with avector such that the vector is internalized by the cell, wherein thevector comprises: (i) a coding sequence located between first and secondrecombining sites in antiparallel orientations and adjoining the secondrecombining site such that the coding sequence is oriented to betranscribed in the opposite direction from the direction of the firstrecombining site to the second recombining site proceeding through thecoding sequence, and wherein the coding sequence is not operably linkedto any promoter, and (ii) a promoter located in a region other than theregion between the first and second recombining sites which comprisesthe coding sequence and adjoining the first recombining site such thatthe promoter is oriented to direct transcription in the direction fromthe first recombining site to the second recombining site proceedingthrough the coding sequence; and (b) providing the cell with asite-specific recombinase that effects recombination between the firstand second recombining sites of the vector.

Preferably, to allow for simultaneous up- and down-regulation ofseparate coding sequences, the vector further comprises a second codingsequence located in the region between the first and second recombiningsites which comprises the coding sequence and adjoining the firstrecombining site, and which is operably linked to the promoter.

Furthermore, in another embodiment providing for down-regulation of asingle coding sequence, the method comprises: (a) contacting the cellwith a vector such that the vector is internalized by the cell, whereinthe vector comprises: (i) a coding sequence located between first andsecond recombining sites in antiparallel orientations and adjoining thefirst recombining site such that the coding sequence is oriented to betranscribed in the direction from the first recombining site to thesecond recombining site proceeding through the coding sequence, and (ii)a promoter adjoining the first recombining site and located in a regionother than the region between the first and second recombining siteswhich comprises the coding sequence, such that the promoter is orientedto direct transcription in the direction from the first recombining siteto the second recombining site proceeding through the coding sequence,and wherein the coding sequence is operably linked to the promoter; and(b) providing the cell with a site-specific recombinase that effectsrecombination between the first and second recombining sites of thevector.

Preferably these vectors may further comprise one or more passengergenes. Optimally there are no coding sequences, promoters, ortranscription termination sites located between the promoter and therecombining site, or between the coding sequence or second codingsequence and the recombining site. Furthermore, splice sites (i.e.,splice acceptor sites and splice donor sites) and polyadenylation sitescan be incorporated in the vectors employed in these methods aspreviously described to maximize gene expression and production ofencoded protein.

Some of these embodiments of the antiparallel recombination method aredepicted in FIGS. 5A-5G. In particular, the method of the inventionwherein a vector comprising antiparallel recombination sites (FRS andSRS) is provided to a cell is depicted. Recombinase also preferably isprovided to the cell, either in cis or in trans. The vector (eitherlinear or circular) employed in this method also preferably comprises acoding sequence (CS). When a promoter is located in the region betweenthe first and second recombining sites, the antiparallel recombinationmethod can be employed to up-regulate a coding sequence (FIG. 5A),down-regulate a coding sequence (FIG. 5B), or simultaneously up-regulatea first coding sequence (CS) and down-regulate a second coding sequence(SCS; FIG. 5C) by the recombination event. One or more splice donor (SD)and/or splice acceptor (SA) sites can be incorporated into the vector tooptimize protein production (FIG. 5D).

Regulation of gene expression effected by the recombination eventsimilarly can be accomplished with use of a vector having a promoterlocated in a region other than the ori-containing region between thefirst and second recombining sites (e.g., when a linear substrate isemployed). Such a vector can be employed to up-regulate acoding-sequence (FIG. 5E), down-regulate a coding sequence (FIG. 5F), orsimultaneously up-regulate a first coding sequence and down-regulate asecond coding sequence (FIG. 5G) by the recombination event. Accordingto the invention, any of the coding sequences (i.e., first, second, orany other coding sequence) and/or any passenger gene present in thevector can encode an EBNA-1, Rep, Flp, Cre, large T antigen, E1, or E2coding sequence. Further variations of this method will be apparent toone of skill in the art.

Novel Vectors

While, as described above with respect to the present inventive methods,there are a variety of vectors which can be used in the context of thepresent invention, the present invention also provides certain novelvectors which are particularly useful in the present inventive paralleland antiparallel recombination methods.

The present inventive vector comprises first and second recombiningsites, located in parallel or in antiparallel orientation. The first andsecond recombining sites can be any suitable recombining sites suchthat, after internalization by a cell and contact with a suitablerecombinase, recombination will occur between the first and secondrecombining sites of the vector.

With respect to recombining sites, recombinase, coding sequences,regions between recombining sites, promoters, passenger genes, and thelike, located on all vectors in the context of the present invention,such elements are as previously described and can be present as part ofa cassette, either independently or coupled. In the context of thepresent invention, a "cassette" is simply a particular base sequencethat possesses functions which facilitate subcloning and recovery ofnucleic acid sequences (e.g., one or more restriction sites) orexpression (e.g., polyadenylation or splice sites) of particular nucleicacid sequences.

Vector identification and/or selection can be accomplished using avariety of approaches known to those skilled in the art. For instance,vectors containing particular genes or coding sequences can beidentified by hybridization, the presence or absence of so-called"marker" gene functions, and the expression of particular insertedsequences. In the first approach, the presence of a foreign sequenceinserted in a vector can be detected by hybridization (e.g., by DNA-DNAhybridization) using probes comprising sequences that are homologous tothe inserted nucleic acid sequence. In the second approach, therecombinant vector/host system can be identified and selected based uponthe presence or absence of certain marker gene functions such asresistance to antibiotics, thymidine kinase activity, and the like,caused by the insertion of particular genes encoding these functionsinto the vector. In the third approach, vectors can be identified byassaying for the foreign gene product expressed by the inserted nucleicacid sequence. Such assays can be based on the physical, immunological,or functional properties of the gene product.

The present inventive vectors which are particularly useful in theparallel and antiparallel recombination methods are further describedbelow.

a. Parallel Recombination Vector

In one embodiment, the present inventive parallel recombination vectorcomprises: (a) first and second recombining sites located in parallel,(b) an origin of replication located between the first and secondrecombining sites, (c) a coding sequence in the region between the firstand second recombining sites which comprises the origin and adjoiningthe first recombining site such that the coding sequence is oriented tobe transcribed in the direction from the first recombining site to thesecond recombining site proceeding through the coding sequence, thecoding sequence is not operably linked to any promoter, and (d) apromoter located in the region between the first and second recombiningsites which comprises the origin and adjoining the second recombiningsite such that the promoter is oriented to direct transcription in thedirection from the first recombining site to the second recombining siteproceeding through the promoter. Preferably there are no codingsequences, promoters, or transcription termination sites located betweenthe first recombining site and the coding sequence.

Furthermore, preferably the vector further comprises a second codingsequence located in a region other than the region between the first andsecond recombining sites which comprises the origin and adjoining thesecond recombining site, and which is operably linked to the promoter.Optimally there are no coding sequences, promoters, or transcriptiontermination sites located between the promoter and the second codingsequence. The vector further can comprise additional coding sequences,for instance a third coding sequence as previously described.

To optimize gene expression and protein production, the vectorpreferably further comprises: (a) a splice acceptor site located betweenthe first recombining site and the coding sequence in the region betweenthe first and second recombining sites which comprises the origin, and(b) a splice donor site located between the promoter and the secondrecombining site in the region between the first and second recombiningsites which comprises the origin, and (c) a splice acceptor site locatedbetween the second recombining site and the second coding sequence in aregion other than that including the region between the first and secondrecombining sites which comprises the origin. Such a vector provides ameans of modulating gene expression by providing an episome in which thefirst coding sequence is up-regulated and the second coding sequence isdown-regulated by the episome-forming recombination event.

In yet another embodiment, the present inventive parallel recombinationvector comprises: (a) first and second recombining sites located inparallel, (b) an origin of replication located between the first andsecond recombining sites, (c) a promoter located in the region betweenthe first and second recombining sites which comprises the origin andadjoins the second recombining site such that the promoter is orientedto direct transcription in the direction from the first recombining siteto the second recombining site proceeding through the promoter, and (d)a coding sequence located in a region other than the region between thefirst and second recombining sites which comprises the origin andadjoining the second recombining site such that the coding sequence isoperably linked to the promoter.

The recombining sites within the parallel recombination vector can beany suitable recombination sites as described above with respect to thepresent inventive methods. Preferably, the recombining sites within theparallel recombination vector are Lox sites, is in which event theparallel recombination vector preferably further comprises the codingsequence for Cre either upstream of the first Lox site or downstream ofthe second Lox site, or the recombining sites within the parallelrecombination vector are Frt sites, in which event the parallelrecombination vector preferably further comprises the coding sequencefor Flp either upstream of the first Frt site or downstream of thesecond Frt site.

The origin of replication is as previously described and preferablycomprises an EBV oriP sequence, a replication origin of polyomavirus orpapillomavirus, or any other replication origin. With use of these (aswell as other) replication origins, preferably further proteins thatenable or facilitate cell replication can be provided to the cell. Thesefurther proteins, that in some sense can be considered "cell replicationproteins", preferably are selected from the group consisting of theEBNA-1, large T antigen, E1, and E2 proteins.

The coding sequence can be any suitable coding sequence. Preferably, thefirst coding sequence is an EBNA-1 or Rep coding sequence, as describedwith respect to the present inventive methods. However, the first codingsequence (and the second coding sequence, or any other coding sequence)also preferably can comprise a Flp, Cre, large T antigen, E1, and/or E2coding sequence. Such a vector provides a means of modulating geneexpression by providing an episome in which the coding sequence isregulated by the episome-forming recombination event.

Preferably, the vector further comprises a passenger gene, which ispreferably located between the coding sequence and the promoter. Anysuitable passenger gene can be employed, such as a reporter gene or,most preferably, a therapeutic gene, as described in the context of thepresent inventive methods. Moreover, the invention provides furthervectors as previously described and as illustrated in the accompanyingFigures.

b. Antiparallel Recombination Vector

The present inventive antiparallel recombination vector is a vectorcomprising: (a) a promoter located between first and second recombiningsites in antiparallel orientations and adjoining the second recombiningsite such that the promoter is oriented to direct transcription in thedirection from the first recombining site to the second recombining siteproceeding through the promoter, and (b) a coding sequence located in aregion other than the region between the first and second recombiningsites which comprises the promoter and adjoining the first recombiningsite such that the coding sequence is oriented to be transcribed in theopposite direction from the direction of the first recombining site tothe second recombining site proceeding through the promoter, wherein thecoding sequence is not operably linked to any promoter.

Preferably the vector further comprises a second coding sequence locatedin a region other than the region between the first and secondrecombining sites which comprises the promoter and adjoining the secondrecombining site, and which is operably linked to the promoter.Optimally there are no coding sequences, promoters, or transcriptiontermination sites located between the first recombining site and thefirst coding sequence, or between the promoter and the second codingsequence.

Moreover, in another embodiment the present invention provides a vectorcomprising: (a) a promoter located between first and second recombiningsites in antiparallel orientations and adjoining the second recombiningsite such that the promoter is oriented to direct transcription in thedirection from the first recombining site to the second recombining siteproceeding through the promoter, and (b) a coding sequence located in aregion other than the region between the first and second recombiningsites which comprises the promoter and adjoining the second recombiningsite such that the coding sequence is operably linked to the promoter.

To optimize gene expression and protein production, the antiparallelrecombination vector further comprises: (a) a splice acceptor sitelocated between the first recombining site and the coding sequence in aregion other than that including the region between the first and secondrecombining sites which comprises the promoter, (b) a splice donor sitelocated between the promoter and the second recombining site in theregion between the first and second recombining sites which comprisesthe promoter, and (c) a splice acceptor site located between the secondrecombining site and the second coding sequence in a region other thanthat including the region between the first and second recombining siteswhich comprises the promoter.

Moreover, the present invention also provides an antiparallelrecombination vector comprising: (a) a coding sequence located betweenfirst and second recombining sites in antiparallel orientations andadjoining the second recombining site such that the coding sequence isoriented to be transcribed in the opposite direction from the directionof the first recombining site to the second recombining site proceedingthrough the coding sequence, and wherein the coding sequence is notoperably linked to any promoter, and (b) a promoter located in a regionother than the region between the first and second recombining siteswhich comprises the coding sequence and adjoining the first recombiningsite such that the promoter is oriented to direct transcription in thedirection from the first recombining site to the second recombining siteproceeding through the coding sequence.

Preferably the vector further comprises a second coding sequence locatedin the region between the first and second recombining sites whichcomprises the coding sequence and adjoining the first recombining site,and which is operably linked to the promoter. The vector also preferablycomprises other coding sequences (e.g., a third coding sequence, and/orother coding sequence), as previously described.

Furthermore, in another embodiment the vector comprises: (a) a codingsequence located between first and second recombining sites inantiparallel orientations and adjoining the first recombining site suchthat the coding sequence is oriented to be transcribed in the directionfrom the first recombining site to the second recombining siteproceeding through the coding sequence, and (b) a promoter adjoining thefirst recombining site and located in a region other than the regionbetween the first and second recombining sites which comprises thecoding sequence, such that the promoter is oriented to directtranscription in the direction from the first recombining site to thesecond recombining site proceeding through the coding sequence, andwherein the coding sequence is operably linked to the promoter.

Preferably these vectors may further comprise a passenger gene.Optimally there are no coding sequences, promoters, or transcriptiontermination sites located between the promoter and the recombining site,or between the coding sequence or second coding sequence and therecombining site. Furthermore, splice sites (i.e., splice acceptor sitesand splice donor sites) and polyadenylation sites can be incorporated inthe vectors employed in these methods as previously described tomaximize gene expression and production of encoded protein.

Such a vector according to the invention provides a means of modulatinggene expression without the generation of an episome as a consequence ofthe recombinase-driven recombination event. Further vectors aspreviously described and depicted in the accompanying Figures also areprovided by the present invention.

Other General Considerations

In the methods of the present invention, in terms of provision of thevarious aspects of the invention to cells, vectors are transferred to ahost cell, which is preferably a eukaryotic host cell. The eukaryotichost cell can be present in vitro or in vivo. According to theinvention, "contacting" of cells with the vectors of the presentinvention can be by any means by which the vectors will be introducedinto the cell. Preferably the viral vectors will be introduced byinfection using the natural capability of the virus to enter cells(e.g., the capability of adenovirus to enter cells via receptor-mediatedendocytosis). However, the viral and plasmid vectors can be introducedby any other suitable means, e.g., transfection, calciumphosphate-mediated transformation, microinjection, electroporation,osmotic shock, and the like. Similarly, in a preferred embodiment of thepresent invention, in vivo transfer of vectors is contemplated.Accordingly, the method of the present invention also contemplatesvector transfer in vivo by the methods set forth herein, or by anystandard method.

Also, according to the methods of the present invention, "providing thecell with a site-specific recombinase" encompasses providing therecombinase encoded by a vector, or providing the recombinase in theform of protein. Introduction of protein can be effected by any meansappropriate for protein introduction to a cell, e.g., microinjection andadenoviral-mediated uptake for in vitro delivery, and injection orinfusion or further means as described herein for in vivo delivery, aswell as by other standard means of introduction known by those skilledin the art.

When practiced in vivo, any suitable organs or tissues or componentcells can be targeted for vector or protein delivery. Preferably, theorgans/tissues/cells employed are of the circulatory system (i.e.,heart, blood vessels or blood), respiratory system (i.e., nose, pharynx,larynx, trachea, bronchi, bronchioles, lungs), gastrointestinal system(i.e., mouth, pharynx, esophagus, stomach, intestines, salivary glands,pancreas, liver, gallbladder), urinary system (i.e., kidneys, ureters,urinary bladder, urethra), nervous system (i.e., brain and spinal cord,and special sense organs such as the eye) and integumentary system(i.e., skin). Even more preferably the cells being targeted are selectedfrom the group consisting of heart, blood vessel, lung, liver,gallbladder, urinary bladder, and eye cells.

For these embodiments, when one or more vectors are employed in themethods described herein, or when recombinase such as Cre or Frt isadministered exogenously in the form of protein, the contacting of cellswith the various components of the present invention can occur in anyorder or can occur simultaneously. Preferably the contacting will occursimultaneously. In a preferred embodiment, the component vectors of thepresent invention can be mixed together and preincubated prior tocontacting the cell. When multiple vectors are to be administered,preferably the cell is contacted with the first vector less than about 6weeks after, or less than about 6 weeks before, the cell is contactedwith another vector. Even more preferably the cell is contacted with thefirst vector less than about 2 weeks after, or less than about 2 weeksbefore, the cell is contacted with another vector. When vectors are tobe administered in combination with recombinase such as Cre or Frt,preferably the cell is contacted with the protein less than about 12hours after, or less than about 12 hours before, the cell is contactedwith a vector. Even more preferably the cell is contacted with theprotein less than about 12 hours after, or less than about 12 hoursbefore, the cell is contacted with a vector.

The composition of the present invention (i.e., a composition comprisingthe vectors and/or recombinases of the present invention) can be madeinto a pharmaceutical composition with appropriate pharmaceuticallyacceptable carriers or diluents, and where appropriate, can beformulated into preparations in solid, semi-solid, liquid or gaseousforms such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, and aerosols, in theusual ways for their respective route of administration. Means known inthe art can be utilized to prevent release and absorption of thecomposition until it reaches the target organ, tissue, or cell, or toensure timed-release of the composition. A pharmaceutically acceptableform should be employed which does not ineffectuate the composition ofthe present invention. In pharmaceutical dosage forms, the compositioncan be used alone or in appropriate association, as well as incombination with, other pharmaceutically active compounds.

Accordingly, the pharmaceutical composition of the present invention canbe delivered via various routes and to various sites in an animal bodyto achieve a particular effect. Local or systemic delivery can beaccomplished by administration comprising application or instillation ofthe formulation into body cavities, inhalation or insufflation of anaerosol, or by parenteral introduction, comprising intramuscular,intravenous, peritoneal, subcutaneous, intradermal, as well as topicaladministration.

The composition of the present invention can be provided in unit dosageform wherein each dosage unit, e.g., a teaspoonful, tablet, solution, orsuppository, contains a predetermined amount of the composition, aloneor in appropriate combination with other active agents. The term "unitdosage form" as used herein refers to physically discrete units suitableas unitary dosages for human and animal subjects, each unit containing apredetermined quantity of the composition of the present invention,alone or in combination with other active agents, calculated in anamount sufficient to produce the desired effect, in association with apharmaceutically acceptable diluent, carrier, or vehicle, whereappropriate. The specifications for the novel unit dosage forms of thepresent invention depend on the particular effect to be achieved and theparticular pharmacodynamics associated with the pharmaceuticalcomposition in the particular host.

Accordingly, the present invention also provides a method of obtainingstable gene expression in a host, or modulating gene expression in ahost, which comprises administering the composition of the presentinvention using any of the aforementioned routes of administration oralternative routes known to those skilled in the art and appropriate fora particular application. The "effective amount" of the composition issuch as to produce the desired effect in a host which can be monitoredusing several end-points known to those skilled in the art. For example,one desired effect might comprise effective nucleic acid transfer to ahost cell. Such transfer could be monitored in terms of a therapeuticeffect (e.g. alleviation of some symptom associated with the disease orsyndrome being treated), or by further evidence of the transferred geneor coding sequence or its expression within the host (e.g., using thepolymerase chain reaction, Northern or Southern hybridizations, ortranscription assays to detect the nucleic acid in host cells, or usingimmunoblot analysis, antibody-mediated detection, or particularizedassays to detect protein or polypeptide encoded by the transferrednucleic acid, or impacted in level or function due to such transfer).One such particularized assay described in the Examples which followincludes an assay for expression of the β-glucuronidase gene.

These methods described are by no means all-inclusive, and furthermethods to suit the specific application will be apparent to theordinary skilled artisan. Moreover, the effective amount of thecomposition can be further approximated through appropriate assay of therecombination reaction, as previously described.

Generally, to ensure effective transfer of the vectors of the presentinvention, it is preferable that about 1 to about 5,000 copies of thevector be employed per cell to be contacted, based on an approximatenumber of cells to be contacted in view of the given route ofadministration, and even more preferable that about 1 to about 300 pfuenter each cell. However, this is merely a general guideline which by nomeans precludes use of a higher or lower amount of a component, as mightbe warranted in a particular application, Either in vitro or in vivo.For example, the actual dose and schedule can vary depending on whetherthe composition is administered in combination with other pharmaceuticalcompositions, or depending on interindividual differences inpharmacokinetics, drug disposition, and metabolism. Similarly, amountscan vary in in vitro applications depending on the particular cell typeutilized or the means by which the vector is transferred. One skilled inthe art easily can make any necessary adjustments in accordance with thenecessities of the particular situation.

EXAMPLES

The following examples further illustrate the present invention but, ofcourse, should not be construed as in any way limiting its scope.

Example 1

This example describes methods and vectors for site-specificrecombination in a cell, and which can be employed to generate anepisome, as set forth in FIGS. 1A-1C. In particular, this exampledemonstrates the method of episome delivery and up-regulation of genetranscription by the recombination event.

Standard molecular and genetic techniques such as generation of strainsand plasmids, gel electrophoresis, DNA manipulations including plasmidisolation, DNA cloning and sequencing, Southern blot assays, and thelike, were performed such as are known to those skilled in the art andas are described in detail in standard laboratory manuals (e.g.,Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (ColdSpring Harbor, N.Y., 1992); Ausubel et al., Current Protocols inMolecular Biology, (1987)). Restriction enzymes and other enzymes usedfor molecular manipulations were purchased from commercial sources(e.g., Boehringer Mannheim, Inc., Indianapolis, Ind.; New EnglandBiolabs, Beverly, Mass.; Bethesda Research Laboratories, Bethesda, Md.;etc.), and were used according to the recommendations of themanufacturer. Cells of the transformed human embryonic kidney cell line293 (American Type Culture Collection CRL 1573) were cultured andmaintained using standard sterile culture reagents, media andtechniques, as previously described (Erzerum et al., Nucleic AcidsResearch, 21, 1607-1612 (1993)). Puromycin was employed, whereappropriate, for election of transfected cells.

Several plasmids were generated to test and confirm the method of thepresent invention as set out in FIGS. 1A-1C. These plasmids includepEOBspLx-Puro-CMVLx, which comprises as the FCS the EBNA-1 codingsequence, and which is depicted in FIG. 6. PlasmidpEOBspLx-Puro-CMVLx-βglu is depicted in FIG. 7, and comprises as the FCSthe β-glucuronidase coding sequence. Both plasmids pEOBspLx-Puro-CMVLxand pEOBspLx-Puro-CMVLx-βglu comprise Lox sites (in parallel) as therecombining sites, and include a CMV promoter as a promoter forup-regulating gene expression. The CMV promoter was employed since it isable to drive a relatively high level of constitutive gene expression inmost tissue culture cells (Boshart et al., Cell, 41, 521-530 (1985)).Similarly, both plasmids pEOBspLx-Puro-CMVLx andpEOBspLx-Puro-CMVLx-βglu comprise the puromycin coding sequence underthe control of the RSV promoter. The Cre-expressing plasmid pAdCMV Cre,which places the phage P1 Cre coding sequence under the control of theCMV promoter and SV40 polyadenylation signal is depicted in FIG. 8.

Plasmid pEOBspLx-Puro-CMVLx was generated from Invitrogen's pREP 4vector (Invitrogen, San Diego, Calif.). pREP 4 was restricted with Hpaland BsmI, and an oligonucleotide containing the restriction sites MluI,PvuII and KpnI was introduced after the unique MluI site in the vectorwas modified. A cassette containing a puromycin gene under the controlof the RSV promoter and bovine growth hormone (BGH) polyadenylationsignal was cloned on a MluI/PvuII DNA fragment into the vector backbone.A CMV promoter containing a splice signal followed by a polylinker and aSV40 polyadenylation sequence was introduced into the PvuII/KpnI sitesof the vector backbone. A Lox site was introduced into the intron of theCMV expression cassette. Sequences upstream of the EBNA-1 codingsequence were modified by the addition of a synthetic splice acceptorsite and Lox site. The splice acceptor site is proximal to EBNA-1 inpEOBspLx-Puro-CMVLx.

Plasmid pEOBspLx-Puro-CMVLx-βglu is derived from pEOBspLx-Puro-CMVLx byreplacing the coding sequence for EBNA-1 with the β-glucuronidase codingsequence and SV40 polyadenylation signal. The resultant deletion in theEBNA-1 coding sequence spans the region from the initiation codon to theEBNA-1 BsgI site.

Plasmid pAdCMV Cre contains an expression cassette comprising the CMVpromoter, Cre coding sequence and SV40 polyadenylation site flanked byAds sequences 1-355 and 3333-5788. Of course, the precise limits of thesequences employed for recombination can vary (e.g., from about 1 toabout 100 bp) depending on the particular recombination product desired.The pAdCMV Cre plasmid can be recombined with Ad to generate anE1-deficient virus (see, e.g., Rosenfeld et al. (1991), supra; Rosenfeldet al. (1992), supra).

To test the ability to select for cells containingpEOBspLx-Puro-CMVLx-βglu by selecting for puromycin resistance impartedby the RSV/puromycin cassette, CaPO₄ -mediated transfections into EBNA-1expressing-293 cells (293-EBNA; InVitrogen, San Diego, Calif.) ofpEOBspLx-Puro-CMVLx-βglu were carried out. Following transfection, cellswere maintained in the presence of puromycin at a concentration of about0.5 mg/ml. The surviving cell population was puromycin resistant due topuromycin expression from the RSV/puromycin cassette.

CaPO₄ -mediated transfections into 293 cells of pEOBspLx-Puro-CMVLx-βgluwith or without pAdCMV Cre were carried out (Graham et al., J. Gen.Virol., 36, 59-72 (1977)) to determine whether Cre recombinase producedby the Cre gene supplied in trans to 293 cells could effectrecombination between Lox sites present in pEOBspLx-Puro-CMVLx-βglu,thus generating an episome and placing the β-glucuronidase codingsequence in the episome under the control of the CMV promoter. After 48hours, the cells were processed and assayed for β-glucuronidase activityusing a commercial kit according to the recommendations of themanufacturer (Tropix Inc., Bedford, Mass.). The obtained results are setforth in Table 2.

                  TABLE 2                                                         ______________________________________                                        Levels of β-glucuronidase following                                      transfection.                                                                 Plasmid(s)             β-glucuronidase units                             ______________________________________                                        No Plasmid             850                                                    pECBspLx-Puro-CMvLx-βglu                                                                        6.9 × 10.sup.3                                   pEOBspLx-Puro-CMvLx-βglu + pAdCMV Cre                                                           1.6 × 10.sup.5                                   ______________________________________                                    

As set forth in Table 2, β-glucuronidase levels are highest in thosecells containing both pEOBspLx-Puro-CMVLx-βglu and pAdCMV Cre. In theabsence of both pEoBspLx-Puro-CMVLx-βglu and pAdCMV Cre (i.e., the "noplasmid" condition), β-glucuronidase levels are barely detectible in 293cells, although a low basal level of activity is observed. Incomparison, a higher level of β-glucuronidase is observed inpEOBspLx-Puro-CMVLx-βglu-containing cells in the absence of pAdCMV Cre.

Subsequently, it was discovered that the recombining sites inpEOBspLx-Puro-CMVLx-βglu are in an antiparallel orientation instead ofthe parallel orientation. Thus, the β-glucuronidase activity observedostensibly was due to the presence of a cryptic promoter placed upstreamof the reporter gene by the recombination event. However, these resultsdo confirm that the method of the invention can be employed to obtain anantiparallel recombination vector.

Example 2

This example describes vectors for site-specific recombination in acell, and which can be employed to generate an episome, as set forth inFIGS. 1A-1C. More particularly, this example describes such vectorswhich are linear Ad vectors.

The method of the present invention similarly can be employed for viralepisome delivery vectors, particularly Ad episome delivery vectors,through modification of the vectors and approach described in the priorexample. Namely, the functionality of oriP, or a similar origin ofreplication capable of functioning in mammalian cells, in such a viralvector can be confirmed by transfecting pEOBspLx-Puro-CMVLx-βglu into acell line that constitutively expresses EBNA-1 (such as described inReisman et al., supra). Cells resistant to puromycin can be selected andexpanded. Southern blot analysis can be employed to confirm themaintenance of the plasmid as an episome. The functionality of EBNA-1can be similarly confirmed.

To generate the Ad vectors, both the EBNA-1 and β-glucuronidase-basedplasmids can be transferred into a shuttle vector and constructed in asimilar fashion as pAdCMV Cre. Basically, the shuttle vector comprises astuffer fragment flanked by BclI and BamHI restriction sites surroundedon one side by the left 355 bp, and on the other side by residues3333-5788 from Ads. The stuffer fragment was replaced with the BglII toBamHI restriction fragment harboring both Lox sites frompEOBspLx-Puro-CMVLx generating plasmid pAdER-Puro. Similarly, thestuffer fragment can be replaced with the BglII to BamHI restrictionfragment harboring both Lox sites from pEOBspLx-Puro-CMVLx-βglu. Eitherof these plasmids can then be used to generate Ad5-based vectors byhomologous recombination as previously described (see, e.g., Rosenfeldet al. (1991), supra; Rosenfeld et al. (1992), supra).

Example 3

This example describes further vectors that can be employed foreffecting site-specific recombination in a cell. In particular, thisexample describes a representative example of a vector that can beemployed to provide Cre recombinase.

To facilitate vector construction, and particularly the vector depictedin FIGS. 1A-1C, the Cre gene was subcloned into a separate Ad. P1obtained from ATCC was used as the source of the Cre recombinase codingsequence, which was cloned into pCR-Script™ (Stratagene, La Jolla,Calif.) using PCR to provide a more accessible source of the gene. TheCre gene was then transferred into a shuttle vector in which it wasplaced under the control of the CMV promoter, and followed by a SV40polyadenylation signal to generate vector pAdCMVCreRSVOrf6 depicted inFIG. 9. The shuttle vector was used to generate the Ad vector AdCMVCre6(using the methods previously described), in which the Cre gene replacesthe E1 region of the adenoviral genome.

Production of AdCMVCre6 was confirmed by PCR and with use of afunctional assay as described in Example 1. Namely, Southern blotanalysis of AdCMVCre6-directed recombination was validated with use of atest plasmid in both the 293-based cell line 835, as well as in thehuman lung carcinoma cell line A549. Similarly, the functioning of thepromoter, splice donor, and splice acceptor sites used in the variousconstructs of this and the previous Examples also were confirmed. Inparticular, β-glucuronidase was placed in position of the first codingsequence in FIG. 1A to generate the plasmid pEOBglugl. Cotransfection ofpEOBglugl with a Cre-expressing plasmid resulted in more than a 3-loginduction of β-glucuronidase.

These results confirm the functionality of Cre recombinase in thisvector, and further confirm the functionality of the base vectorcomponents used for subsequent vector constructions.

Example 4

This example describes further vectors for use for effectingsite-specific recombination in a cell. In particular, this exampledescribes vectors that comprise a polyomavirus origin of replication andthat can be used, for instance, to obtain runaway replication.

The vectors containing the polyomavirus origin were constructed usingstandard cloning techniques as described in the previous Examples. Thefunctioning of the components of the EBV and polyomavirus systemscontained in the various vectors and required for replication (inparticular, the origin of replication) was confirmed with use of a Dpn Iassay. The basis of this assay is that plasmids produced in E. coli aremethylated and are sensitive to restriction by Dpn I. In comparison, ifthe plasmid replicates in mammalian cells, it either is hemimethylated,or is not methylated at all, which renders it resistant to Dpn Irestriction.

The isogenic constructs pKSEBNAOriP(NR) (depicted in FIG. 10) andpKSEBNA(NS) (depicted in FIG. 11) were tested to confirm functionalityof the EBV component. These plasmids contain EBNA-1 under the control ofits own viral promoter in either the presence (i.e., plasmidpKSEBNAOriP(NR)) or absence (i.e., plasmid pKSEBNA(NS)) of oriP locatedimmediately 5' of the gene. The oriP origin in pKSEBNAOri(NR) has adeletion from Nco I to Eco RV (i.e., EBV coordinates 8033 to 8995),resulting in a 962 bp deletion. The plasmids were transfected into293-derived cells and harvested 5 days later. The cell pellets weresubjected to Hirt extraction (Hirt, J. Mol. Biol., 26, 365-369 (1967)),and the Dpn I assay was performed. Only the oriP-containing plasmidswere found to replicate.

Similarly, the plasmid pAdCLxPyTagOri (depicted in FIG. 12) wastransfected into NIH 3T3 cells. This plasmid contains the polyomaviruslarge T antigen coding sequence under the control of the CMV promoter,and contains the polyomavirus origin immediately 3' of thepolyadenylation signal flanking the T antigen coding sequence. A similarplasmid containing these elements except for the T antigen was found toreplicate when T antigen was provided to the cell. These results thusconfirm that the polyomavirus components present in this vector, likethe EBV components present in pKSEBNAOriP(NR), is capable of impartingautonomous replication on a plasmid in which it is present.

Following confirmation of the Functioning of the polyomaviruscomponents, the cassette present in pAdCLxPyTagOri was transferred intothe Ad E1 deletion shuttle vector pADEPrRLr depicted in FIG. 13. Thisplasmid is essentially as described in FIG. 1A with the first codingsequence (FCS) comprising the T antigen coding sequence and the operatorbeing the polyomavirus origin of replication. The promoter (P) in thisplasmid is an RSV promoter, and the second coding sequence (SCS)comprises a luciferase reporter gene. The activity of the luciferasetranscriptional unit was confirmed. Moreover, transfection of thisplasmid with a Cre expression cassette resulted in site-specificrecombination of the shuttle vector. These results thus confirm thefunctionality of the various components of the vectors.

The results validate that other vectors for use for effectingsite-specific recombination in a cell can be constructed and employedaccording to the invention. In particular, the results confirm thatvectors that comprise a polyomavirus origin of replication can beconstructed and used, for instance, to obtain runaway replication.

Example 5

This example describes further vectors for use for effectingsite-specific recombination in a cell. In particular, this exampledescribes vectors that comprise a bovine papillomavirus origin ofreplication and that can be used for obtaining a multicopy number in awide variety of hosts.

Similar techniques as described in the earlier examples were employed toconstruct vectors comprising the bovine papillomavirus origin ofreplication. In particular, the vector pCGE1E2ori dEpicted in FIG. 14was constructed. This vector comprises a dicistronic expression cassettewith the bovine papilloma virus E1 and E2 genes under the control of aCMV promoter. Immediately, 3' of the expression cassette, the vectorcomprises the bovine papillomavirus origin of replication spanningnucleotides 7351-57 of the bovine papillomavirus genome. Anotherconstruct harboring the bovine papillomavirus origin previously wasreported to be stably maintained in the presence of E1 and E2, whichsupports the replication competency of the pCGE1E2ori vector.

All of the references cited herein, including patents, patentapplications, and publications, are hereby incorporated in theirentireties by reference.

While this invention has been described with an emphasis upon preferredembodiments it will be apparent to those of ordinary skill in the artthat variations in the preferred embodiments can be prepared and usedand that the invention can be practiced otherwise than as specificallydescribed herein. The present invention is intended to include suchvariations and alternative practices. Accordingly, this inventionincludes all modifications encompassed within the spirit and scope ofthe invention as defined by the following claims.

What is claimed is:
 1. A vector comprising:(i) a first recombining siteand a second recombining site in parallel orientation, and (ii) one ormore adeno-associated viral ITRs, wherein said vector optionally furthercomprises a passenger gene.
 2. The vector of claim 1, which furthercomprises:(i) a promoter, which (a) is located in the region betweensaid first and second recombining sites, (b) adjoins said secondrecombining site, and (c) is oriented to direct transcription in thedirection from said first recombining site to said second recombiningsite proceeding through said promoter, and (ii) a coding sequence, whichis located in the region between said first and second recombiningsites, which comprises said promoter, adjoins said first recombiningsite, is oriented to be transcribed in the direction from said firstrecombining site to said second recombining site proceeding through saidpromoter, and is not operably linked to said promoter.
 3. A method ofeffecting site-specific recombination in a eukaryotic cell, which methodcomprises:(a) contacting said cell with a vector of claim 2, such thatsaid vector is internalized by said cell, and (b) providing said cellwith a site-specific recombinase that effects recombination between saidfirst and second recombining sites of said vector, wherein, uponrecombination, said coding sequence is operably linked to said promoter.4. The vector of claim 1, which further comprises:(i) a promoter, which(a) is located in the region between said first and second recombiningsites, (b) adjoins said second recombining site, and (c) is oriented todirect transcription in the direction from said first recombining siteto said second recombining site proceeding through said promoter, and(ii) a coding sequence, which is located in a region other than theregion between said first and second recombining sites, which comprisessaid promoter, adjoins said second recombining site, and is operablylinked to said promoter.
 5. A method of effecting site-specificrecombination in a eukaryotic cell, which method comprises:(a)contacting said cell with a vector of claim 4, such that said vector isinternalized by said cell, and (b) providing said cell with asite-specific recombinase that effects recombination between said firstand second recombining sites of said vector, wherein, uponrecombination, said coding sequence is not operably linked to saidpromoter.
 6. The vector of claim 1, which further comprises:(i) apromoter, which (a) is located in the region between said first andsecond recombining sites, (b) adjoins said first recombining site, and(c) is oriented to direct transcription in the direction from said firstrecombining site to said second recombining site proceeding through saidpromoter, and (ii) a coding sequence, which is located in a region otherthan the region between said first and second recombining sites, whichcomprises said promoter, adjoins said second recombining site, isoriented to be transcribed in the direction from said first recombiningsite to said second recombining site proceeding through said promoter,and is operably linked to said promoter.
 7. A method of effectingsite-specific recombination in a eukaryotic cell, which methodcomprises:(a) contacting said cell with a vector of claim 6, such thatsaid vector is internalized by said cell, and (b) providing said cellwith a site-specific recombinase that effects recombination between saidfirst and second recombining sites of said vector, wherein, uponrecombination, said coding sequence is not operably linked to saidpromoter.
 8. The vector of claim 1, which further comprises:(i) apromoter, which is (a) located in a region other than the region betweensaid first and second recombining sites, (b) adjoins said firstrecombining site, and (c) is oriented to direct transcription in thedirection from said promoter to said second recombining site proceedingthrough said first recombining site, and (ii) a coding sequence, whichis located in the region between said first and second recombiningsites, adjoins said first recombining site, and is operably linked tosaid promoter.
 9. A method of effecting site-specific recombination in aeukaryotic cell, which method comprises:(a) contacting said cell with avector of claim 8, such that said vector is internalized by said cell,and (b) providing said cell with a site-specific recombinase thateffects recombination between said first and second recombining sites ofsaid vector, wherein, upon recombination, said coding sequence is notoperably linked to said promoter.
 10. The vector of claim 1, whereinsaid coding sequence, first coding sequence, second coding sequence orpassenger gene comprises a sequence encoding a ligand binding domain ofa hormone receptor that is capable of binding an exogenous, but not anaturally-occurring, hormone.
 11. The vector of claim 1, wherein saidcoding sequence, first coding sequence, second coding sequence orpassenger gene comprises one or more sequences encoding aglycine-alanine repeat.
 12. A vector comprising:(i) a first recombiningsite and a second recombining site in parallel orientation, (ii) anorigin of replication and/or one or more adeno-associated viral ITRs,(iii) one or more promoters, and (iv) one or more coding sequences,wherein, if said origin of replication is present, it is located betweensaid first and second recombining sites, wherein, each of said one ormore promoters and each of said one or more coding sequences adjoins thefirst or second recombining site, such that one or more of said firstand second recombining sites is/are adjoined by one or more promotersand/or one or more coding sequences, wherein, upon recombination,upregulation of one or more coding sequence(s) and/or downregulation ofone or more other coding sequence(s) is/are effected, and wherein saidvector optionally further comprises a passenger gene.
 13. The vector ofclaim 12, which comprises:(i) a promoter, which (a) is located in theregion between said first and second recombining sites, which, if thevector comprises an origin, comprises said origin, (b) adjoins saidsecond recombining site, and (c) is oriented to direct transcription inthe direction from said first recombining site to said secondrecombining site proceeding through said promoter, and (ii) a codingsequence, which is located in the region between said first and secondrecombining sites, which comprises said promoter, adjoins said firstrecombining site, is oriented to be transcribed in the direction fromsaid first recombining site to said second recombining site proceedingthrough said promoter, and is not operably linked to said promoter. 14.A method of effecting site-specific recombination in a eukaryotic cell,which method comprises:(a) contacting said cell with a vector of claim13, such that said vector is internalized by said cell, and, if thevector comprises an origin of replication, the origin of replication isfunctional in a mammalian cell, and (b) providing said cell with asite-specific recombinase that effects recombination between said firstand second recombining sites of said vector, wherein, uponrecombination, said coding sequence is operably linked to said promoter.15. The vector of claim 12, which comprises:(i) a promoter, which (a) islocated in the region between said first and second recombining sites,which, if the vector comprises an origin, comprises said origin, (b)adjoins said second recombining site, and (c) is oriented to directtranscription in the direction from said first recombining site to saidsecond recombining site proceeding through said promoter, and (ii) acoding sequence, which is located in a region other than the regionbetween said first and second recombining sites, which comprises saidpromoter, adjoins said second recombining site, and is operably linkedto said promoter.
 16. A method of effecting site-specific recombinationin a eukaryotic cell, which method comprises:(a) contacting said cellwith a vector of claim 15, such that said vector is internalized by saidcell, and, if the vector comprises an origin of replication, the originof replication is functional in a mammalian cell, and (b) providing saidcell with a site-specific recombinase that effects recombination betweensaid first and second recombining sites of said vector, wherein, uponrecombination, said coding sequence is not operably linked to saidpromoter.
 17. The vector of claim 12, which comprises:(i) a promoter,which (a) is located in the region between said first and secondrecombining sites, which, if the vector comprises an origin, comprisessaid origin, (b) adjoins said first recombining site, and (c) isoriented to direct transcription in the direction from said firstrecombining site to said second recombining site proceeding through saidpromoter, and (ii) a coding sequence, which is located in a region otherthan the region between said first and second recombining sites, whichcomprises said promoter, adjoins said second recombining site, isoriented to be transcribed in the direction from said first recombiningsite to said second recombining site proceeding through said promoter,and is operably linked to said promoter.
 18. A method of effectingsite-specific recombination in a eukaryotic cell, which methodcomprises:(a) contacting said cell with a vector of claim 17, such thatsaid vector is internalized by said cell, and, if the vector comprisesan origin of replication, the origin of replication is functional in amammalian cell, and (b) providing said cell with a site-specificrecombinase that effects recombination between said first and secondrecombining sites of said vector, wherein, upon recombination, saidcoding sequence is not operably linked to said promoter.
 19. The vectorof claim 12, which comprises:(i) a promoter, which is (a) located in aregion other than the region between said first and second recombiningsites, which, if the vector comprises an origin, comprises said origin,(b) adjoins said first recombining site, and (c) is oriented to directtranscription in the direction from said promoter to said secondrecombining site proceeding through said first recombining site, and(ii) a coding sequence, which is located in the region between saidfirst and second recombining sites, adjoins said first recombining site,and is operably linked to said promoter.
 20. A method of effectingsite-specific recombination in a eukaryotic cell, which methodcomprises:(a) contacting said cell with a vector of claim 19, such thatsaid vector is internalized by said cell, and, if the vector comprisesan origin of replication, the origin of replication is functional in amammalian cell, and (b) providing said cell with a site-specificrecombinase that effects recombination between said first and secondrecombining sites of said vector, wherein, upon recombination, saidcoding sequence is not operably linked to said promoter.
 21. The vectorof claim 12, wherein said coding sequence, first coding sequence, secondcoding sequence or passenger gene comprises a sequence encoding a ligandbinding domain of a hormone receptor that is capable of binding anexogenous, but not a naturally-occurring, hormone.
 22. The vector ofclaim 12, wherein said coding sequence, first coding sequence, secondcoding sequence or passenger gene comprises one or more sequencesencoding a glycine-alanine repeat.
 23. A method of effectingsite-specific recombination in a eukaryotic cell, which methodcomprises:(a) contacting said cell with a vector of claim 12, such thatsaid vector is internalized by said cell, and, if the vector comprisesan origin of replication, the origin of replication is functional in amammalian cell, and (b) providing said cell with a site-specificrecombinase that effects recombination between said first and secondrecombining sites of said vector.
 24. A vector comprising a firstrecombining site and a second recombining site in parallel orientation,between which are located (i) a first coding sequence, which adjoinssaid first recombining site such that said first coding sequence isoriented to be transcribed in the direction from said first recombiningsite to said second recombining site proceeding through said firstcoding sequence and which is not operably linked to a promoter, and (ii)a promoter, which adjoins said second recombining site such that saidpromoter is oriented to direct transcription in the direction from saidfirst recombining site to said second recombining site proceedingthrough said promoter,wherein said vector optionally further comprises apassenger gene, an origin of replication located in the region betweensaid first and second recombining sites comprising said first codingsequence, one or more adeno-associated viral ITRs, said passenger geneand said origin, or said passenger gene and said one or moreadeno-associated viral ITRs.
 25. The vector of claim 24, wherein saidvector further comprises a second coding sequence, which is located in aregion other than the region between said first and second recombiningsites comprising said first coding sequence and which adjoins saidsecond recombining site such that said second coding sequence isoperably linked to said promoter.
 26. The vector of claim 25, whereinsaid vector further comprises:(i) a splice acceptor site, which islocated between said first recombining site and said first codingsequence such that splice acceptor site is in the region between saidfirst and second recombining sites comprising said first codingsequence, (ii) a splice donor site, which is located between saidpromoter and said second recombining site such that said splice donorsite is in the region between said first and second recombining sitescomprising said first coding sequence, and (iii) a splice acceptor site,which is located between said second recombining site and second codingsequence such that said splice acceptor site is in a region other thanthe region between said first and second recombining sites comprisingsaid first coding sequence.
 27. A method of effecting site-specificrecombination in a eukaryotic cell, which method comprises:(a)contacting said cell with a vector of claim 26, and (b) providing saidcell with a site-specific recombinase that effects recombination betweensaid first and second recombining sites of said vector.
 28. A method ofeffecting site-specific recombination in a eukaryotic cell, which methodcomprises:(a) contacting said cell with a vector of claim 25, and (b)providing said cell with a site-specific recombinase that effectsrecombination between said first and second recombining sites of saidvector, wherein, upon recombination, said second coding sequence is notoperably linked to said promoter.
 29. The vector of claim 24, whereinsaid vector further comprises:(i) a second coding sequence, which islocated in a region other than the region between said first and secondrecombining sites, which comprises said first coding sequence and saidpromoter, adjoins said first recombining site such that said secondcoding sequence is oriented to be transcribed in the opposite directionfrom the direction of said first recombining site to said secondrecombining site proceeding through said first coding sequence, and isnot operably linked to a promoter, and (ii) a second promoter, which islocated in a region other than the region between said first and secondrecombining sites, which comprises said first coding sequence, andadjoins said second recombining site such that said second promoter isoriented to direct transcription in the opposite direction from thedirection of said first recombining site to said second recombining siteproceeding through said first coding sequence.
 30. A method of effectingsite-specific recombination in a eukaryotic cell, which methodcomprises:(a) contacting said cell with a vector of claim 29, and (b)providing said cell with a site-specific recombinase that effectsrecombination between said first and second recombining sites of saidvector,wherein, upon recombination, said first coding sequence isoperably linked to said first promoter and said second coding sequenceis operably linked to said second promoter.
 31. The vector of claim 24,wherein said coding sequence, first coding sequence, second codingsequence or passenger gene comprises a sequence encoding a ligandbinding domain of a hormone receptor that is capable of binding anexogenous, but not a naturally-occurring, hormone.
 32. The vector ofclaim 24, wherein said coding sequence, first coding sequence, secondcoding sequence or passenger gene comprises one or more sequencesencoding a glycine-alanine repeat.
 33. A method of effectingsite-specific recombination in a eukaryotic cell, which methodcomprises:(a) contacting said cell with a vector of claim 24, and (b)providing said cell with a site-specific recombinase that effectsrecombination between said first and second recombining sites of saidvector, wherein, upon recombination, said first coding sequence isoperably linked to said promoter.
 34. A vector comprising:(i) a firstrecombining site and a second recombining site in parallel orientationbetween which is located a first coding sequence, which adjoins saidfirst recombining site and is oriented to be transcribed in thedirection from said first recombining site to said second recombiningsite proceeding through said first coding sequence, (ii) a promoter,which is located in a region other than the region between said firstand second recombining sites, which comprises said first codingsequence, and adjoins said first recombining site such that saidpromoter is operably linked to said first coding sequence, and (iii) asecond coding sequence, which is located in a region other than theregion between said first and second recombining sites, which comprisessaid first coding sequence, adjoins said second recombining site, and isoriented to be transcribed in the direction from said first recombiningsite to said second recombining site proceeding through said firstcoding sequence, wherein said vector optionally further comprises apassenger gene, an origin of replication located in the region betweensaid first and second recombining sites comprising said first codingsequence, one or more adeno-associated viral ITRs, said passenger geneand said origin, or said passenger gene and said one or moreadeno-associated viral ITRs.
 35. The vector of claim 34, wherein saidcoding sequence, first coding sequence, second coding sequence orpassenger gene comprises a sequence encoding a ligand binding domain ofa hormone receptor that is capable of binding an exogenous, but not anaturally-occurring, hormone.
 36. The vector of claim 34, wherein saidcoding sequence, first coding sequence, second coding sequence orpassenger gene comprises one or more sequences encoding aglycine-alanine repeat.
 37. A method of effecting site-specificrecombination in a eukaryotic cell, which method comprises:(a)contacting said cell with a vector of claim 27, and (b) providing saidcell with a site-specific recombinase that effects recombination betweensaid first and second recombining sites of said vector,wherein, uponrecombination, said first coding sequence is not operably linked to saidpromoter and said second coding sequence is operably linked to saidpromoter.
 38. A vector comprising a first recombining site and a secondrecombining site in antiparallel orientation and one or moreadeno-associated viral ITRs, wherein said vector optionally furthercomprises a passenger gene.
 39. The vector of claim 38, which furthercomprises one or more promoters and one or more codingsequences,wherein, each of said one or more promoters and each of saidone or more coding sequences adjoins the first or second recombiningsite, such that one or both of said first and second recombining sitesis/are adjoined by one or more promoters and/or one or more codingsequences, and wherein, upon recombination, upregulation of one or morecoding sequence(s) and/or downregulation of one or more other codingsequence(s) is/are effected.
 40. The vector of claim 39, wherein saidvector comprises:(i) a promoter, which is located in the region betweensaid first and second recombining sites, adjoins said second recombiningsite, and is oriented to direct transcription in the direction from saidfirst recombining site to said second recombining site proceedingthrough said promoter, and (ii) either a coding sequence "a", which islocated in a region other than the region between said first and secondrecombining sites comprising said promoter, adjoins said firstrecombining site, is oriented to be transcribed in the oppositedirection from the direction of said first recombining site to saidsecond recombining site proceeding through said promoter, and is notoperably linked to a promoter, or a coding sequence "b", which islocated in a region other than the region between said first and secondrecombining sites comprising said promoter, adjoins said secondrecombining site, and is operably linked to said promoter.
 41. A methodof effecting site-specific recombination in a cell comprising:(a)contacting said cell with a vector of claim 40, such that said vector isinternalized by said cell, and (b) providing said cell with asite-specific recombinase that effects recombination between said firstand second recombining sites of said vector,wherein, upon recombination,either said coding sequence "a" is operably linked to said promoter orsaid coding sequence "b" is not operably linked to said promoter. 42.The vector of claim 39, wherein said vector comprises:(i) a promoter,which is located in a region other than the region between said firstand second recombining sites, adjoins said first recombining site and isoriented to direct transcription in the direction from said promoter tosaid second recombining site proceeding through said first recombiningsite, and (ii) either a coding sequence "a", which is located in theregion between said first and second recombining sites, adjoins saidfirst recombining site and is operably linked to said promoter, or acoding sequence "b", which is located in the region between said firstand second recombining sites, adjoins said second recombining site, isoriented to be transcribed in the opposite direction from the directionof said first recombining site to said second recombining siteproceeding through said coding sequence, and is not operably linked tosaid promoter.
 43. A method of effecting site-specific recombination ina cell comprising:(a) contacting said cell with a vector of claim 42,such that said vector is internalized by said cell, and (b) providingsaid cell with a site-specific recombinase that effects recombinationbetween said first and second recombining sites of said vector,wherein,upon recombination, either said coding sequence "a" is not operablylinked to said promoter or said coding sequence "b" is operably linkedto said promoter.
 44. A method of effecting site-specific recombinationin a cell comprising:(a) contacting said cell with a vector of claim 39,such that said vector is internalized by said cell, and (b) providingsaid cell with a site-specific recombinase that effects recombinationbetween said first and second recombining sites of said vector.
 45. Thevector of claim 38, wherein said coding sequence, first coding sequence,second coding sequence or passenger gene comprises a sequence encoding aligand binding domain of a hormone receptor that is capable of bindingan exogenous, but not a naturally-occurring, hormone.
 46. The vector ofclaim 38, wherein said coding sequence, first coding sequence, secondcoding sequence or passenger gene comprises one or more sequencesencoding a glycine-alanine repeat.
 47. A method of effectingsite-specific recombination in a cell comprising:(a) contacting saidcell with a vector of claim 38, such that said vector is internalized bysaid cell, and (b) providing said cell with a site-specific recombinasethat effects recombination between said first and second recombiningsites of said vector.
 48. A vector comprising:(i) a first recombiningsite and a second recombining site in anti-parallel orientation betweenwhich is located a promoter, which adjoins said second recombining siteand is oriented to direct transcription in the direction from said firstrecombining site to said second recombining site proceeding through saidpromoter, (ii) a first coding sequence, which is in a region other thanthe region between said first and second recombining sites, whichcomprises said promoter, adjoins said first recombining site, isoriented to be transcribed in the opposite direction from the directionof said first recombining site to said second recombining siteproceeding through said promoter, and is not operably linked to saidpromoter, (iii) a second coding sequence, which is in a region otherthan the region between said first and second recombining sites, whichcomprises said promoter, adjoins said second recombining site, and isoperably linked to said promoter, and (iv) (a) one or moreadeno-associated viral ITRs, (b) a passenger gene, or (c) one or moreadeno-associated viral ITRs and a passenger gene.
 49. The vector ofclaim 48, wherein said coding sequence, first coding sequence, secondcoding sequence or passenger gene comprises a sequence encoding a ligandbinding domain of a hormone receptor that is capable of binding anexogenous, but not a naturally-occurring, hormone.
 50. The vector ofclaim 48, wherein said coding sequence, first coding sequence, secondcoding sequence or passenger gene comprises one or more sequencesencoding a glycine-alanine repeat.
 51. A method of effectingsite-specific recombination in a cell comprising:(a) contacting saidcell with a vector of claim 48, such that said vector is internalized bysaid cell, and (b) providing said cell with a site-specific recombinasethat effects recombination between said first and second recombiningsites of said vector,wherein, upon recombination, said second codingsequence, which was operably linked to said promoter, is not operablylinked to said promoter, and said first coding sequence, which was notoperably linked to said promoter, is operably linked to said promoter.52. A vector comprising:(i) a first recombining site and a secondrecombining site in anti-parallel orientation between which are locateda first coding sequence, which adjoins said second recombining site andis oriented to be transcribed in the opposite direction from thedirection of said first recombining site to said second recombining siteproceeding through said first coding sequence, and a second codingsequence, which adjoins said first recombining site and is oriented tobe transcribed in the direction of said first recombining site to saidsecond recombining site proceeding through said second coding sequence,(ii) a promoter, which is in a region other than the region between saidfirst and second recombining sites, which comprises said first andsecond coding sequences, adjoins said first recombining site, and isoperably linked to said second coding sequence, and (iii) (a) one ormore adeno-associated viral ITRs, (b) a passenger gene, or (c) one ormore adeno-associated viral adeno-associated viral ITRs and a passengergene.
 53. The vector of claim 52, wherein said coding sequence, firstcoding sequence, second coding sequence or passenger gene comprises asequence encoding a ligand binding domain of a hormone receptor that iscapable of binding an exogenous, but not a naturally-occurring, hormone.54. The vector of claim 52, wherein said coding sequence, first codingsequence, second coding sequence or passenger gene comprises one or moresequences encoding a glycine-alanine repeat.
 55. A method of effectingsite-specific recombination in a eukaryotic cell, which methodcomprises:(a) contacting said cell with a vector of claim 52, such thatsaid vector is internalized by said cell, and (b) providing said cellwith a site-specific recombinase that effects recombination between saidfirst and second recombining sites of said vector.
 56. A method ofeffecting site-specific recombination in a eukaryotic cellcomprising:(a) contacting said cell with a vector comprising:(i) a firstrecombining site and a second recombining site in parallel orientation,and (ii) an origin of replication, which is located between said firstand second recombining sites and is functional in a mammalian cell,and/or one or more adeno-associated viral ITRs, wherein said vector isinternalized by said cell, and (b) providing said cell with asite-specific recombinase that effects recombination between said firstand second recombining sites of said vector.
 57. A vector comprising:(i)a first recombining site and a second recombining site, (ii) one or moreadeno-associated viral ITRs, and (iii) minimal sequences required forpackaging and delivery of the vector to a cell.
 58. The vector of claim57, wherein the minimal sequences required for packaging and delivery ofthe vector to a cell are derived from an adenovirus.
 59. The vector ofclaim 58, wherein said vector is an adenoviral vector.
 60. The vector ofclaim 57, wherein the vector further comprises a nucleic acid sequenceencoding all or part of a Rep protein.
 61. The vector of claim 57,wherein the vector comprises at least one passenger gene.
 62. The vectorof claim 61, wherein the vector further comprises regulatory sequencesrequired for expression of the at least one passenger gene.